Methods of inhibiting transmission of a costimulatory signal of lymphocytes

ABSTRACT

Novel cell surface molecules recognized by monoclonal antibodies against a cell surface molecule of lymphocytic cells that play an important role in autoimmune diseases and allergic diseases have been isolated, identified, and analyzed for their functions. The cell surface molecules are expressed specifically in thymocytes, lymphocytes activated by ConA-stimulation, and peripheral blood lymphocytes, and induce cell adhesion. Antibodies against the cell surface molecules significantly ameliorate pathological conditions of autoimmune diseases and allergic diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation and claims the benefit of priorityunder 35 USC §120 of U.S. application Ser. No. 09/561,308, filed Apr.28, 2000 now U.S. Pat. No. 7,112,655, which is a divisional of U.S.application Ser. No. 09/383,551, filed Aug. 26, 1999 now U.S. Pat. No.7,030,225, which is a continuation-in-part of international applicationnumber PCT/JP98/00837, filed Feb. 27, 1998, which claims the benefit ofpriority under 35 USC §119 of Japanese application number 9/62290, filedFeb. 27, 1997, and Japanese application number 10/62217, filed Feb. 26,1998. The disclosures of the prior applications are considered part of(and are incorporated by reference in) the disclosure of thisapplication.

TECHNICAL FIELD

The present invention relates to novel cell surface molecules ofmammals; polypeptides and their fragments constituting the molecules;fusion polypeptides comprising the polypeptide fragments andimmunoglobulin fragments; genes encoding the polypeptides and thefragments; vectors comprising the genes; transformants into which thevectors are introduced; antibodies having reactivity to the polypeptidesor cell surface molecules comprising the polypeptides; hybridomasproducing the antibodies; pharmaceutical compositions comprising thepolypeptide fragments or the fusion polypeptides; pharmaceuticalcompositions comprising the antibodies; transgenic mice; and knockoutmice.

BACKGROUND ART

A living body of mammals has immune response systems that excludespathogenic microorganisms (viruses, bacteria, parasites, etc.) orforeign bodies (both are called “antigen” in the following) that haveinvaded the living body. One of them is called natural immune responsesystem, another acquired immune response system. The former is anexclusion mechanism comprising phagocytosis by phagocytes(polymorphonuclear leukocytes, monocytes, macrophages, etc.), attack bynatural killer (NK) cells, and non-specific recognition such asopsonization of antigen by complements. The latter, acquired immuneresponse system, is an exclusion mechanism by lymphocytes (mainly, Tcells and B cells) that acquired the specificity to the antigen (namely,activated lymphocytes). B cells that acquired antigen specificityattacks the antigen existing outside of the cells through production ofantibodies specific to the antigen. T cells that acquired antigenspecificity (namely, activated T cells) are classified into helper Tcells and cytotoxic T cells (cytotoxic lymphocyte, CTL). The helper Tcells regulate a differentiation of B cells and a production ofantibodies, and destroy the antigen cooperating with phagocytes. Thelatter, CTLs attack virus-infected cells and so on by themselves(Experimental Medicine: SUPPLEMENT, “Bio Science Term Library,Immunity”, Yodosha, pp. 14-17 (1995)).

This acquisition of antigen specificity by T cells (namely, activationof T cells) is initiated through recognition by T cells the antigenpresented by antigen-presenting cells (APC) such as macrophage, B cells,or dendritic cells. Antigen-presenting cells process the antigens soincorporated and present these processed antigens through binding themto major histocompatibility complex (MHC). T cells receives primarysignal for activation of the cells (or acquisition of specificity) byrecognizing the processed antigens presented by antigen-presenting cellsthrough a complex between T cell receptor (TcR) and CD3 antigen existingon the surface of the cell membrane (TcR/CD3 complex).

However, the TcR/CD3 complex-mediated primary signal alone cannotactivate T cells sufficiently and leads to unresponsiveness or clonalanergy, so that the cells can not react with any stimulation receivedthereafter. The autocrine of interleukin 2 (IL-2) is necessary for Tcells to be activated, to be differentiated into antigen specific T cellclones, and to be proliferated. In clonal anergy, T cells areinactivated due to no production of IL-2 and no cell division. Namely,the activation of T cells accompanied by production of cytokines such asIL-2 requires the secondary signal following the first signal throughTcR/CD3 complex. This secondary signal is called costimulatory signal.

T cells receive this secondary signal and transmit it into the cells byinteracting (cell adhesion) with molecules other than MHC onantigen-presenting cells through other molecules other than TcR/CD3complex on the T cell surface. This secondary signal avoids cell anergy(clonal anergy) and activates the cells.

Although some part of the mechanism of the secondary signal transmissionbetween antigen-presenting cells and lymphocytes such as T cells havenot yet been elucidated in detail, studies so far have revealed that animportant factor for the secondary signal transmission is theinteraction of CD28 (also named Tp44, T44, or 9.3 antigen), which is acell surface molecule expressed mainly on T cells and thymus cells, withCD80 (also named B7-1, B7, BB1, or B7/BB1), which is a cell surfacemolecule expressed on antigen-presenting cells (macrophages, monocytes,dendritic cells, and so on etc.) and with CD86 (also named B7-2 or B70),which is also a cell surface molecule on antigen-presenting cells(namely, cell adhesion through the binding between these molecules).Moreover, it has been experimentally elucidated that the interaction ofCytolytic T lymphocyte associated antigen 4 (CTLA-4), whose expressionis thought to be enhanced depending on the secondary signal, with theCD80 (B7-1) and CD86 (B7-2) (namely, cell adhesion through the bindingbetween these molecules) also plays an important role in the regulationof T cell activation by the secondary signal. In other words, theregulation of T cell activation by the transmission of the secondarysignal involves, at least the interaction between CD28 and CD80/CD86,the enhancement of CTLA-4 expression, which is thought to depend on theinteraction, and the interaction between CTLA-4 and CD80/CD86.

CD28 is known to be a costimulator molecule transmitting the secondarysignal (costimulatory signal) required for the activation of T cells andfor the avoidance of anergy. The secondary signal transmitted by bindingthis molecule to costimulator molecules, CD80 (B7-1) and CD86 (B7-2), onantigen-presenting cells (namely, cell adhesion through the bindingbetween these molecules), stabilizes mRNA of Th1-type cytokines andconsequently promotes production by T cells of a large amount ofproduction of Th1-type cytokines such as Il-2, IFNγ, and TNFα. Theexpression of CTLA-4 is induced by the primary signal transmittedthrough TcR/CD3, and the expression is also enhanced by the secondarysignal transmitted by the binding between CD28 and CD80. It is beingrevealed that CTLA-4 receives these signals to work to inhibit T cellfunction, which is contrary to the activation of T cells by thesecondary signal transmitted by CD28.

Human CD28 and CTLA-4 are I-type glycoproteins whose molecular weightsare 44 kD and 41 to 43 kD, respectively. Both have animmunoglobulin-like domain, belong to the immunoglobulin superfamily,and have both function as a cell adhesion molecule and function as asignal transmission molecule.

Human CD28 forms a homodimer with a disulfide bond while CTLA-4 existsas a monomer. Both CD28 and CTLA-4 genes are located at “2q33” on humanchromosome and “1C” on mouse chromosome, and are composed of four (4)exons. Human CD28 and CTLA-4 are composed of 220 and 223 amino acids,respectively, including the leader sequences, and amino acid homologybetween them is 20 to 30%.

The ligands for CD28 and CTLA-4 are CD80 (B7-1) and CD86 (B7-2) in humanand mice. CTLA-4 has about 20 as higher affinity to both ligands asCD28. It has been elucidated that the amino acid sequence structures“MYPPPY (Met-Tyr-Pro-Pro-Pro-Tyr)” (SEQ ID NO:18 conserved throughanimal species is important for the binding of CD28 and CTLA-4 to CD80(B7-1). It has also been reported that, when CD28 is stimulated, PI3kinase (phosphoinositide 3 kinase, PI3K) associates with thephosphorylated tyrosine residue in a partial sequence “YMNM(Tyr-Met-Asn-Met)” (SEQ ID NO:19) and that CD28 play an important rolein intracellular signal transmission through this “YxxM” structure.Furthermore, it has been reported that CTLA-4 also has a sequencerepresented by “YxxM,” namely “YVKM (Tyr-Val-Lys-Met)” (SEQ ID NO:20)inits cytoplasmic region and that, after being stimulated, SYP associateswith this sequence.

CD28 is expressed specifically in thymocytes and peripheral blood Tcells, and CTLA-4 is expressed specifically in activated T cells (CellEngineering: SUPPLEMENT, “Handbook of Adhesion Molecule”, Shujunsha, pp.93-102 (1994); ibid. pp. 120-136; Experimental Medicine: SUPPLEMENT,“BIO SCIENCE Term Library, Immunity”, Yodosha, pp. 94-98 (1995);Experimental Medicine: SUPPLEMENT, “BIO SCIENCE Term Library,Intracellular Signal Transduction”, Yodosha, pp. 58-59 (1997); NihonRinsho, Vol.55, No.6, pp. 215-220 (1997)).

In the regulation of T cell function (the activation and the inhibitionof function of T cells), the importance of interactions among multiplemolecules such as costimulator molecules (CD28, CD80 (B7-1), CD86(B7-2), etc.) and CTLA-4, which cooperates with them, (in other words,cell adhesion through the binding between these molecules) has thus beenrecognized, and this has been drawn attention to the relationshipbetween these molecules and diseases, and the treatment of diseases byregulating the function of these molecules have been noted.

As described above, although a living body activates its acquired immuneresponse system against antigens that are foreign bodies to the livingbody (self), it also has immunological tolerance so as to show no immuneresponse against its own component (autoantigen). If immunologicaltolerance breaks down by some reason, immune response to the autoantigenoccurs, autoantigen-reactive T cells are induced by the same mechanismas mentioned above to fall into abnormal state of immunity, and variousautoimmune diseases are caused.

In other words, since non-stimulated antigen presenting cells (APC) innormal tissues do not express costimulatory molecules when the immunesystem of a living body is normal, T cells fall are in theunresponsiveness state to maintain immunological tolerance even ifautoantigen-reactive T cells, which reacts with autoantigen, exist. Ithas been suggested that in abnormal state of immunity, moreautoantigen-reactive T cells are activated due to abnormal excess andcontinuous expression of costimulatory molecules to thereby causeautoimmune diseases.

From such viewpoints recently, many attempts to treat for variousautoimmune diseases by modulating the transmission of costimulatorysignals, for example, the above-mentioned signal transmission betweenCD28/CTLA-4 and CD80/CD86, are proposed.

It has been observed CD80, a costimulatory molecule as the ligand ofCD28 and CTLA-4, is abnormally expressed in the antigen presenting cellsat the nidus of autoimmune disease such as rheumatoid arthritis,multiple sclerosis, autoimmune thyroiditis, allergic contact-typedermatitis, and chronic inflammatory dermatosis such as squamous lichenplanus, and psoriasis. From such observation, many attempts to treatvarious autoimmune diseases by modulating the function of CD80 have beenmade.

It has been proposed to block the function of CD80, by methods using anantibody against CD80, solubilized protein of CD28 that is a ligand ofCD80, and solubilized protein of CTLA-4 that is also a ligand of CD80.Particularly, based on the fact that the binding affinity of CTLA-4 toCD80 is 20 or more times higher than that of CD28, therapeutic attemptsusing “solubilized CTLA-4,” specifically, the fusion protein(CTLA-4-IgFc) comprising the extracellular domain of “CTLA-4” and the Fcregion of human immunoglobulin IgG1, were performed in animal model andclinical tests (Nihon Rinsho, Vol. 55, No. 6, pp. 215-220 (1997)).

As shown in 1 to 5 below, therapeutic effects of CTLA-4-IgFc in modelanimals of autoimmune diseases has been reported.

1. In a (NZB/NZW)F1 mouse, that is a model for human systemic lupuserythematosus (SLE), the production of autoantibodies and the onset oflupus nephritis were suppressed by administration of CTLA-4-IgFc beforethe onset, and the pathologic conditions were improved by administrationof the drug even after the onset (Science, Vol. 125, p. 1225-1227(1994)).

2. In experimental allergic encephalomyelitis (EAE), that is a model formultiple sclerosis (MS), the onset was prevented by short-termadministration of CTLA-4-IgFc immediately after immunization (J. Clin.Invest., Vol.95, pp. 2783-2789 (1995)).

3. In an NOD (non-obese diabetes) mouse, which is a model for insulindependent diabetes mellitus (IDDM), the onset rate was remarkablydecreased by administering CTLA-4-IgFc to the 2- or 3-week-old femalemouse for two weeks (J. Exp. Med. 181:1145-1155, 1995).

4. In rat nephritis by renal glomerulus basement membrane immunity,Goodpasture's nephritis model, the improvement of the symptom has beenimproved by the administration of CTLA-4-IgFc (Eur. J. Immunol.24:1249-1254, 1994).

5. In type II collagen-induced arthritis (CIA) using a DBA/1 mouse, thatis a model for human rheumatoid arthritis, the onset of arthritis wassuppressed by the administering the test drug at the time ofimmunization and the production of autoantibodies (IgG1 and IgG2)against collagen was inhibited (Eur. J. Immunol. 26:2320-2328, 1996).

The results of the experiments as mentioned above are have not yetclarified in detail the mechanism of the T cell activation byinteraction between costimulatory molecules and the related molecules(in other words, cell adhesion through the binding between thesemolecules). Other unknown molecules may be involved in this mechanism.

DISCLOSURE OF THE INVENTION

Pharmaceuticals useful for treating or preventing various diseases suchas the above-mentioned autoimmune diseases, allergic diseases, andinflammatory diseases can be developed if the mechanism of theactivation of lymphocytes such as T cells by cell adhesion through thebinding between molecules involved in the transmission of the secondarysignal essential for the activation of lymphocytes such as T cellsmentioned above and the mechanism of the regulation of lymphocytefunction are clarified, and known or unknown molecules capable ofmediating cell adhesion involved in the mechanism and of transmittingsignals are identified and characterized.

An objective of the present invention is to identify novel cell surfacemolecules having both functions of mediating such cell adhesion andsignal transmission, and to clarify its structural and biologicalcharacteristics. Another objective of the present invention is toprovide pharmaceuticals useful for treating or preventing variousautoimmune diseases and inflammatory diseases by using the novelmolecules or antibodies against the molecules.

In order to identify such useful molecules, the present inventorsfocused on the fact that lymphocytes such as T cells play an importantrole in autoimmune diseases, and the fact that cell adhesion areessential for the signal transmission of the secondary signal(costimulatory signal) from antigen presenting cells into lymphocytes,and planned to isolate and identify cell surface molecules that areexpressed specifically on lymphocytic cells and that mediate celladhesion.

The present inventors obtained monoclonal antibodies against variouscell surface molecules expressed on the surface of lymphocytic cells byimmunizing animals against the lymphocytic cells, and isolated andidentified desired cell surface molecules that mediate cell adhesionusing the monoclonal antibodies so obtained. The methods used aredescribed in detail below.

The present inventors first administered rat lymphocytic cell line as anantigen to mice and prepared various monoclonal antibodies. Then, themonoclonal antibodies obtained were reacted with rat lymphocytic cellsused as an antigen and tested the effect of the monoclonal antibodiesgiven to the cells. As a result, one of the monoclonal antibodies wasobtained has been found to agglutinate the rat lymphocytic cellsstrongly (this monoclonal antibody was designated “JTT-1 antibody”).Moreover, other one of the monoclonal antibodies was found to stronglyinhibit the agglutination of rat lymphocytic cells induced by the “JTT-1antibody” (this monoclonal antibody was designated “JTT.2 antibody”).

Since the agglutination of rat lymphocytic cells by “JTT-1 antibody” wasnot inhibited by antibodies against Intercellular adhesion molecule-1(ICAM-1) or Lymphocyte function-associated antigen-1 (LFA-1), which arethe most representative known cell adhesion molecules expressed on thecells, the present inventors thought that this agglutination was causedby cell adhesion through unknown adhesion molecules having that mediatecell adhesion.

Cell surface molecules (designated “JTT-1 antigen” and “JTT.2 antigen”)recognized by each of these two monoclonal antibodies were thenidentified, isolated, and characterized.

First, the analysis of the expression patterns of “JTT-1 antigen” and“JTT.2 antigen” in various cells were analyzed by flow cytometry basedon fluorescent antibody technique using “JTT-1 antibody” and “JTT-2antibody.” While both “JTT-1 antigen” and “JTT.2 antigen” were stronglyexpressed in activated lymphoblast cells (activated T lymphoblast cells,activated B lymphoblast cells, etc.) activated by stimulating thymocytesand spleen cells with Concanavalin A (ConA), a mitogen, in particular,in the activated lymphoblast cells, the expression was hardly found inspleen cells not stimulated at all (these cells are sometimes called“resting lymphocytes” herein). The expression patterns of moleculesrecognized by each of “JTT-1 antibody” and “JTT-2 antibody” were almostthe same.

Using an affinity column prepared by binding “JTT-1 antibody” toadsorbents, molecules trapped by the “JTT-1 antibody”, namely, “JTT-1antigens” were purified from the mixture of soluble cell surfacemolecules prepared from the above-described rat lymphocytic cells. Themolecular weights of these purified “JTT-1 antigens” were analyzed byimmunoprecipitation using “JTT-1 antibody” and “JTT-2 antibody” and bySDS-PAGE. As a result, it was found that molecules immunoprecipitated byeach of “JTT-1 antibody” and “JTT-2 antibody” were the same, and thateach molecule was a homodimer having different sugar chains.Specifically, when N-linked sugar chains were not digested, themolecules were identified as one molecule with about 47 kD undernon-reduction condition, and as two molecules with about 24 kD and about28 kD under reduction condition; and when N-linked sugar chains weredigested, the molecules were identified as one molecule with about 36 kDunder non-reduction condition and as one molecule with about 20 kD underreduction condition.

The adhesion of rat thymocytes to the plate coated by the purified“JTT-1 antigen” was then analyzed. As a result, thymocytes significantlyadhered to the plate (namely, to “JTT-1 antigen”) only in the presenceof “JTT-1 antibody” and that the adhesion was significantly inhibited inthe co-presence of “JTT.2 antibody”, indicating that “JTT-1 antigen” wasthe cell surface molecule mediating cell adhesion.

Next, the present inventors cloned genes encoding “JTT-1 antigen” fromrat, human, and mouse, and analyzed their structures.

First, the cDNA encoding the full length of “rat JTT-1 antigen” wasisolated from the cDNA library made from the lymphoblasts derived fromConA-stimulated rat spleen by expression cloning method utilizingpanning method using “JTT-1 antibody” and a completely novel rat genewas isolated and identified by determining its nucleotide sequence bydideoxy method. The cDNA encoding the full length of “human JTT-1antigen” was also isolated from the cDNA library made fromConA-stimulated human peripheral blood lymphoblasts by plaquehybridization with using the cDNA encoding “rat JTT-1 antigen” soobtained as a probe and a completely novel human gene was isolated andidentified by determining its nucleotide sequence by dideoxy method.Similarly, the cDNA encoding the full length of “mouse JTT-1 antigen”was isolated from the cDNA library made from the lymphoblasts derivedfrom ConA-stimulated mouse spleen and a completely novel mouse gene wasisolated and identified by determining its nucleotide sequence bydideoxy method. Furthermore, the cDNA encoding the full length ofalternative splicing variant of “rat JTT-1 antigen” mentioned above wasisolated similarly from the cDNA library made from the rat thymoma cellline and another completely novel rat gene was isolated and identifiedby determining its nucleotide sequence by dideoxy method.

“JTT-1 antigen” was found to be a transmembrane protein (cell surfacemolecule) composed of a signal sequence, an extracellular region havingthe glycosylation site(s), a transmembrane region, and an intracellularregion by hydropathy plot analysis of the amino acid sequence encoded bythe isolated cDNA of “human JTT-1 antigen”. Homology search with knownmolecules revealed that of “JTT-1 antigens” from rat, human, and mousehad no significant homology to any known molecules including celladhesion molecules, indicating that they are novel cell surfacemolecules that mediates cell adhesion.

As the result that of motif search based on the amino acid sequence of“human JTT-1 antigen”, it was found that “human JTT-1 antigen” hadstructural similarity with the above-mentioned “CD28”, a cell surfacemolecule on lymphocytes such as T cells, which transmits costimulatorysignal important for T cell activation through cell adhesion and with“CTLA-4”, a cell surface molecule on lymphocytes such as T cells, whichregulates the functions of activated lymphocytes such as activated Tcells, cooperating with the signal.

The structural similarity is as follows.

1. 20 or more amino acid residues including cysteine residues are highlyconserved.

2. Proline repeating sequence “Pro-Pro-Pro (PPP)” essential as theligand binding region, is conserved in the extracellular region.

3. A sequence “Tyr-Xaa-Xaa-Met (YxxM)” (Xaa and x represents any aminoacid) sequence essential as the signal transmitting region is conservedin the cytoplasmic region.

The locus of the gene encoding “mouse JTT-1 antigen” on mouse chromosomewas found to be “1C3”, which is the same location as that of mouse“CD28” and “CTLA-4” using fluorescence in situ hybridization (FISH)method.

Next, the effectiveness of therapy of autoimmune diseases and allergicdiseases by regulating the function of “JTT-1 antigen”, was examined byexperiments in which “JTT-2 antibody” mentioned above was administeredto model rats for experimental allergic encephalomyelitis (EAE) andglomerulus basement membrane (GBM) nephritis. It was found that thepathological states were significantly suppressed in both disease modelanimals, and that autoimmune diseases or allergic diseases can betreated by regulating the functions of “JTT-1 antigen”.

It was also found that the monoclonal antibody against “human JTT-1antigen” significantly proliferated human peripheral blood lymphocytes,and that the proliferation was further enhanced in the co-presence of amonoclonal antibody against CD3 constituting a TcR/CD3 complex on Tcells, which receives the primary signal essential for T cell activationfrom antigen presenting cells, indicating that “JTT-1 antigen” was acell surface molecule involved in signal transmission into lymphocytes.

Furthermore, the present inventors succeeded in producing a fusionpolypeptide comprising of the extracellular region of “human JTT-1antigen” and Fc region of human immunoglobulin. The fusion polypeptideis useful as pharmaceuticals for treating autoimmune diseases, allergicdiseases, and inflammatory diseases by regulating the “JTT-1 antigen”and/or its ligand.

Moreover, the present inventors succeeded in preparing a transgenicmouse into which a gene encoding “JTT-1 antigen” of other animal specieswas introduced. The transgenic mouse is useful for analyzing detailedfunctions of “JTT-1 antigen” and for developing pharmaceuticals fortreating autoimmune diseases, allergic diseases, and inflammatorydiseases. The inventors also produced a knockout mouse in which theendogenous gene encoding “mouse JTT-1 antigen” was inactivated. Thisknockout mouse is also useful for the above-mentioned purpose.

The present inventions relate to polypeptides, genes, antibodies,vectors, transformants, pharmaceutical compositions, transgenic mice,knockout mice and so on, which are relevant to a novel mammalian “JTT-1antigen” isolated and identified as mentioned above. Specifically, thepresent invention are as described in (1) to (36) below.

(1) A polypeptide constituting a cell surface molecule havingcharacteristics mentioned below,

(a) said cell surface molecule is expressed in at least thymocytes andmitogen-stimulated lymphoblast cells,

(b) an antibody reactive to said cell surface molecule induces adhesionbetween mitogen-stimulated lymphoblast cells,

(c) an antibody reactive to said cell surface molecule inducesproliferation of peripheral blood lymphocytes under the coexistencewithin the presence of an antibody against CD3,

(d) said cell surface molecule has a partial amino acid sequencerepresented by Phe-Asp-Pro-Pro-Pro-Phe (SEQ ID NO:21) in itsextracellular region, and

(e) said cell surface molecule has a partial amino acid sequencerepresented by Tyr-Met-Phe-Met (SEQ ID NO:22) in its cytoplasmic region.

(2) The polypeptide of (1) comprising the amino acid sequence of SEQ IDNO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one or moreamino acids are substituted, deleted, or added.

(3) The polypeptide of (1), which is encoded by a DNA hybridizing with aDNA having the nucleotide sequence of SEQ ID NO: 1 under stringentconditions.

(4) The polypeptide of (1) comprising an amino acid sequence having 60%or more homology with an amino acid sequence of SEQ ID NO: 2.

(5) The polypeptide of any one of (1) to (4) wherein said cell surfacemolecule is derived from human.

(6) A gene encoding the polypeptide of any one of (1) to (5).

(7) The gene of (6) wherein said gene is a cDNA.

(8) The gene of (7) wherein said cDNA has a nucleotide sequence of SEQID NO: 1.

(9) The gene of (7) wherein said cDNA comprises a nucleotide sequencecorresponding to nucleotide residues 26 to 625 of SEQ ID NO: 3,nucleotide residues 35 to 637 of SEQ ID NO: 4, nucleotide residues 1 to603 of SEQ ID NO: 5, or nucleotide residues 35 to 685 of SEQ ID NO: 6.

(10 A vector comprising the gene of any one of (6) to (9).

(11) A transformant into which the vector of (10) has been introduced.

(12) A transformant distinguished identified by an international depositaccession No. FERM BP-5725.

(13) A polypeptide fragment comprising an extracellular region of thepolypeptide of any one of (1) to (5).

(14) The polypeptide fragment of (13) wherein said polypeptide is ahuman-derived polypeptide having an amino acid sequence of SEQ ID NO: 2.

(15) A gene encoding the polypeptide fragment of (13) or (14).

(16) A homodimer molecule comprising two polypeptide fragments, whereineach of the fragments comprises an extracellular region of thepolypeptide of any one of (1) to (5) and said two polypeptide fragmentsbridged through disulfide bonds to each other.

(17) The homodimer molecule of (16) wherein said polypeptide is ahuman-derived polypeptide having an amino acid sequence of SEQ ID NO: 2.

(18) A pharmaceutical composition comprising either of the polypeptidefragment of (14) or the homodimer molecule of (17), or both of them, anda pharmaceutically acceptable carrier.

(19) A fusion polypeptide comprising an extracellular region of thepolypeptide of any one of (1) to (5) and a constant region of a humanimmunoglobulin (Ig) heavy chain or a portion of the constant region.

(20) The fusion polypeptide of (19) wherein the immunoglobulin is IgG.

(21) The fusion polypeptide of (19) wherein the portion of the constantregion comprises a hinge region, C2 domain, and C3 domain of IgG.

(22) The fusion polypeptide of any one of (19) to (21) wherein saidpolypeptide is a human-derived polypeptide having an amino acid sequenceof SEQ ID NO: 2.

(23) A homodimer molecule comprising two fusion polypeptide of any oneof (19) to (22) wherein the two polypeptides bridged through disulfidebonds to each other.

(24) A homodimer molecule comprising two fusion polypeptides of (22)wherein the two polypeptides bridged through disulfide bonds to eachother.

(25) A pharmaceutical composition comprising either of the fusionpolypeptide of (22) or the homodimer molecule of (24), or both of them,and a pharmaceutically acceptable carrier.

(26) The pharmaceutical composition of (25) wherein said pharmaceuticalcomposition is utilized for treating autoimmune diseases or allergicdiseases, or for preventing said disease symptom.

(27) An antibody or a portion thereof reactive to the polypeptide of anyone of (1) to (5), the polypeptide fragment of (13) or (14), or the cellsurface molecule comprising said polypeptide.

(28) The antibody of (27) or a portion of it wherein said antibody is amonoclonal antibody.

(29) An monoclonal antibody or a portion thereof reactive to thepolypeptide having an amino acid sequence of SEQ ID NO: 2, thepolypeptide fragment of (14), or the human-derived cell surface moleculecomprising said polypeptide.

(30) A monoclonal antibody or a portion thereof reactive to thepolypeptide of any one of (1) to (5) or the cell surface moleculecomprising said polypeptide, wherein the effect of said monoclonalantibody on mitogen-stimulated lymphoblast cells is substantially thesame as the effect of a monoclonal antibody produced by a hybridomaidentified by an international deposit accession No. FERM BP-5707 onmitogen-stimulated rat lymphoblast cells.

(31) A monoclonal antibody or a portion thereof reactive to thepolypeptide of any one of (1) to (5) or the cell surface moleculecomprising said polypeptide, wherein the effect of said monoclonalantibody on mitogen-stimulated lymphoblast cells is substantially thesame as the effect of a monoclonal antibody produced by a hybridomaidentified by an international deposit accession No. FERM BP-5708 onmitogen-stimulated rat lymphoblast cells.

(32) A pharmaceutical composition comprising the monoclonal antibody of(29) or a portion thereof and a pharmaceutically acceptable carrier.

(33) The pharmaceutical composition of (32) wherein said pharmaceuticalcomposition is are utilized for treating autoimmune diseases or allergicdiseases, or for preventing said disease symptom.

(34) A hybridoma producing the monoclonal antibody of any one of (28) to(31).

(35) A transgenic mouse in which a gene encoding the polypeptide of (1)which is a human-derived gene comprising a nucleotide sequence of SEQ IDNO: 1 or a rat-derived gene comprising a nucleotide sequencecorresponding to nucleotide residues 35 to 637 of SEQ ID NO: 4, which isintegrated into the mouse its endogenous gene.

(36) A knockout mouse in which its endogenous gene encoding the mousepolypeptide of claim 1 comprising the amino acid sequence encoded by thegene of SEQ ID NO: 5 is inactivated so that said mouse polypeptide isnot produced.

As described above, the cell surface molecule of the present invention(“JTT-1 antigen”) is involved in cell adhesion through the molecule,signal transmission into lymphocytes such as T cells, and functionregulation of function of activated lymphocytes. General knowledge oflymphocytic cells, cell adhesion molecules, and the relationship betweenthem and diseases are described below just for general understanding ofthese biological events but the following general knowledge is not forinterpreting the present invention limitedly.

Lymphocytes are roughly classified into two kinds, T cells and B cells.After differentiation from multipotent stem cells in bone marrow tolymphoid stem cells, some of them flow into blood to reach thymus.Lymphocytes differentiated and matured in thymus, which are called Tcells (Thymus-derived T cells), get into blood again, and circulatethrough the whole body. Matured T cells have a molecule called CD3 ontheir surface. The existence of CD3 molecule is an marker to determinewhether the cells are matured T cells or not. CD3 is a convincing T cellmarker. In addition, T cells express CD4 or CD8. T cells are classifiedinto helper T cells (Th cells) assisting the antibody production by Blymphocytes, cytotoxic T cells (Tc cells, CTL) or killer T cells thatare bound to target cells to destroy them directly, suppressor T cellsthat suppress the antibody production by B lymphocytes, and effector Tcells that secrete effector substances such as lymphokines to causedelayed allergy.

B cells are derived from the lymphoid stem cells differentiated andmatured in bone marrow. B cells are those antibody-producing precursorcells since they produce antibodies with an appropriate stimulus. Bcells have immunoglobulins on their cell surface, which were produced ina cell. Such immunoglobulins function as receptors for antigens. MaturedB cells have both IgM and IgD on their surface. If B cells aredifferentiated with antigen stimulation and signals from T cells, theproduction of IgM increases and their C-terminal cell membrane bindingregions are changed to be secreted. With sufficient stimulation, notonly the surface immunoglobulins change into IgG, IgE, and IgA, but alsothe immunoglobulins of each class are secreted. The immunoglobulin onthe B cell surface is sometimes represented as Ig, abbreviation ofsurface Ig, or mIg, abbreviation of membrane Ig. All Igs on the surfaceof the same B cell have the same antigen binding sites.

There are lymphocytes called large granular lymphocytes (LGL) or nullcells, which are neither T cells nor B cells. These cells can destroytumor cells and virus-infected cells without pre-stimulation withantigen, which is comparative to the case of cytotoxic T cells. So, theyare also called natural killer cells (NK cells).

Among the T cells mentioned above, CD4-positive T cells secrete variouscytokines, newly express receptors for these cytokines, enlarge theirown size, start cell dividing, and proliferate, when they react withantigen-presenting cells. Prior to these reactions at the cell level,the complex between of the antigen peptides on antigen presenting cellsand MHC class II molecules binds to the corresponding T cell antigenreceptor (TCR). This causes various biochemical changes in the cells,and the signal is transmitted into nuclei to start the transcription ofspecific DNAs and to produce respective proteins. As a result, reactionsat the cell level are raised. For example, cells infected with a virusproduce virus proteins and they are degraded into peptides byproteasomes in the cytoplasm. A part of the peptides enters endoplasmicreticulum through TAP, forms stable complex with MHC class I moleculesjust produced, and transfers to the cell surface. The peptidetransferred to the cell surface is recognized specifically byCD8-positive T cells, but the T cells can not yet destroy the infectedcells at this stage. These T cells reacted to with the antigen expressesIL-2 receptor (IL-2R), are differentiated into CTL cellular cytotoxicityupon IL-2 action, and destroy their target cells to kill them in thenext time when they meet the same antigen peptide/MHC class I complex.Cytokines required for the differentiation into CTL are not only IL-2but also IFNγ or other cytokines, which are thought to have similaractions. Thus, lymphokines secreted by T cells are necessary for thedifferentiation into CTL. The lymphokines are produced as the resultthat CD4-positive Th1 cells (CD4-positive T cells secreting IL-2 orINFγ) recognize the antigen peptides derived from the same virus withclass II molecules. In some cases, without the help of CD4-positive Tcells, CD8-positive T cells react with antigens and produce IL-2 andother cytokines. When CD8-positive T cells are differentiated into CTL,granules increase in the cytoplasm. These granules comprise various highmolecular weight proteins, represented by perforin. Perforin resembles amembrane attack complex (MAC) composed of the fifth to ninth componentsof complement, and makes holes in the cell membrane of target cells. Inaddition, the granules comprise serine proteases, LT, and proteoglycan,etc. Moreover, if CD8-positive cells differentiated into CTL receiveantigen stimulation, they also secrete lymphokines such as IFNγ, LT,TNF, or IL-2. Moreover, T cells show blast transformation phenomenon,when they react with hemagglutinin (phytohemagglutinin, PHA) or ConA.

Matured T cells not yet stimulated at all are called resting T cells,and have smaller cell size and shorter lifetime, a few days. When theyreceive stimulation, the cells enlarge as already mentioned above, andare apt to react with various kinds of stimulation. Such T cells arecalled activated T cells. A part of the activated T cells become memoryT cells, which bring secondary immunoreaction if they receive the sameantigen stimulation. Memory T cells are thought to be kept incirculating around the body for a few years or decades.

B cells not yet stimulated at all are called resting B cells like in thecase of T cells, and proliferating B cells stimulated with multivalentantigens or CD40L, are called activated B cells. Since resting B cellshave no costimulator molecules, which stimulate T cells with signalsthrough TCR, such as B7-1 (CD80) or B7-2 (CD86), presenting antigens toresting T cells are thought only to stimulate TCR and to be unable toexpress CD40 ligands (CD40L) or produce lymphokines. Therefore, it isthought that activated helper T cells stimulated with antigen presentedby other antigen-presenting cells react with the antigen presented byresting B cells. Namely, if an antigen invades, first, dendritic cells(cells having extremely dendritic projections) expressing B7 moleculesor macrophages activated by reacting with microorganisms present theantigen and stimulate resting helper T cells to activate them so as toexpress CD40L. The activated helper T cells then bind to resting B cellspresenting the same antigen and stimulate their CD40. Once B cells areactivated by stimulation with multivalent antigens or CD40L, they alsoexpress B7 molecules, activate helper T cells by stimulating CD28 ontheir surface with TCR, and allow the helper T cells to express CD40L orproduce lymphokines. B cells that show changes such as the expansion ofthe cell size with stimulation but not show antibody secretion arecalled activated B cells. If B cells so matured meet antigens, the IgMproduction increases together with the stimulation from T cells and theIgM molecules so produced are secreted by turning from the membrane typeinto secretory type. Moreover, they produce isotypic antibodies otherthan IgM, such as IgG upon the humoral factors from T cells. This iscalled isotype switching or class switching. B cells secretingantibodies are called antibody-secreting cells. A part of them becomesmorphologically characteristic cells and is called a plasma cell(Knowledge of Immunology, Ohmsha, (1996)).

Incidentally, in various reactions of immune system, the subpopulationof white blood cells, namely, T lymphocytes, B lymphocytes, NK,neutrophils, etc., often show dynamics different from one another. Eventhe same lymphocytes as mentioned above show dynamics different from oneanother depending on whether the cells are activated or resting. Thesefacts imply the existence of recognition mechanism specific to thesubpopulation of white blood cells, further, recognition mechanismspecific to the state of cells, and, in particular, cell adhesionmolecules (cell adhesion proteins).

Cell adhesion molecules, namely,or cell adhesion proteins are, ingeneral, the molecules that adhere cells to each other in thedevelopment and differentiation of individuals or in migration of cellsto local site, and are known to be essential molecules for organic andfunctional contacts in a living body.

Cell adhesion molecules are roughly classified from their structuralcharacteristics into five (5) families, immunoglobulin superfamily,integrin family, selectin family, cadherin family, and CD44 family.Adhesion molecules belonging to immunoglobulin superfamily arecharacterized by the existence of repeated loop-like domains formed withdisulfide bonds. Examples thereof are intercellular adhesion molecule-1“ICAM-1” and vascular cell adhesion molecule-1 “VCAM-1.” In addition,adhesion molecules belonging to integrin family are characterized by α/βheterodimer structure. Examples thereof are “VLA-1 to 6” lymphocytefunction-associated antigen-1 “LFA-1”, “Mac-1,” and “p150/90.” Moleculesbelonging to selectin family have lectin-like domain, EGF-like domain,and complement domain in this order from N terminus. Examples thereofare “E-selectin” and “P-selectin.” Examples of cadherin family are“E-cadherin,” “N-cadherin,” and “P-cadherin,” and an example of CD44family is “CD44”.

The specific function of these adhesion molecules is known to beadhesion of white blood cells to vascular endothelial cells or oflymphocytes to antigen-presenting cells. From recent various studies, ithas been gradually revealed that adhesion molecules are involved notonly in these functions but also in various diseases.

In particular, there are many reports on diseases and expressionabnormality of adhesion molecules. For example, as for rheumatoidarthritis (RA), the expression of both “Mac-1” and “p150/95” wasreportedly strengthened in RA synoviocytes (Allen et al., ArthritisRheum., 32:947, 1989). It has also been reported that various cellsexpressed “ICAM-1” strongly and ectopically on RA synovial membrane(Hale et al., Arthritis Rheum., 32:22, 1989). Another report impliedthat “ELAM-1” was also involved in the adhesion of neutrophils tovascular endothelial cells and that the overexpression of thesemolecules was involved in infiltration of neutrophils (especially, intosynovial fluid), which is observed in RA synovial fluid (Laffon et al.,Arthritis Rheum., 32:386, 1989). Strong expression of “CD44” in vascularendothelial cells and A-type synoviocytes on RA synovial membrane wasreported (Heynes et al., Arthritis Rheum., 34:1434, 1991).

There are reports on the relationship between systemic lupuserythematosus (SLE) and the expression abnormality of adhesionmolecules. For example, adhesion ability of T lymphocytes to culturedvascular endothelial cells was reportedly lowered in SLE patients,compared to healthy volunteers. In peripheral lymphocytes of SLEpatients, adhesion molecules “ICAM-1”, “VLA-4”, and “IFA-1” to werestrongly expressed (Haskard et al., Rheumatol. Int., 9:33, 1989).

In autoimmune thyroiditis diseases, it was reported that “ICAM-1” wasexpressed when a thyroid follicular cells were stimulated withinterferon-T, interleukin-1, and tumor necrosis factor, and that theformation of cluster of follicular cells and mononuclear cells wasinhibited by anti-“ICAM-1” antibody (Weetman et al., Eur. J. Immunol.,20:271, 1990).

In hepatitis, it is thought that the chances of adhesion betweenhepatocytes and inflammatory cells increases since there are twopathways of adhesion, “ICAM-1” and “LFA-3”, and “LFA-1” and “CD2”, tothereby promote presentation of antigens and activation of inflammatorycells. In particular, in hepatitis B, “LFA-3” molecules are stronglyexpressed in hepatocytes, in which viruses are actively proliferating,and “ICAM-1” well correlates with the degree of hepatitis. It is thusimplied that “LFA-3” is involved in the exclusion of viruses and“ICAM-1” promotes T cells to present antigen and regulates inflammationreaction. In “ICAM-1”-negative and HBc antigen-positive hepatocytes,chronic virus infection, a kind of immunounresponsiveness, may occur dueto no interaction between lymphocytes and hepatocytes. It has also beenreported that serum “ICAM-1” in chronic liver disease may correlate withthe degree of hepatocyte damage because the serum “ICAM-1”concentrations in acute hepatitis patients, chronic active hepatitispatients, and liver cirrhosis patients were higher than that in healthyvolunteers and chronic persisting hepatitis patients, and theconcentration was high in the case of histologically progressing activehepatitis (Mod. Phys., 15:73-76, 1995).

In a model animal of arteriosclerosis, adhesion and invasion ofmonocytes and lymphocytes to vascular endothelium were observed at veryearly stages of the onset of the disease. It is thus thought that theinteraction of these hemocytes with endothelium is the first step of theonset of arteriosclerosis. Various reports show the expression ofadhesion molecules in actual arteriosclerosis nidus including theexpression of “ICAM-1” in human arteriosclerosis nidus (Poston et al.,Am. J. Pathol., 140:665, 1992) and the expression of “VCAM-1” inarteriosclerosis nidus of a hypercholesterolemia rabbit (Cybulsky etal., Science, 251:788, 1991). A recent report revealed that theexpression of “VCAM-1” was observed in human arteriosclerosis nidus,and, in particular, strong expression in smooth muscle cells migratingto intima and in monocytes/macrophages. In addition, since theexpression of “MCP-1” was enhanced in rabbit and human arteriosclerosisnidus, suggesting that “MCP-1” promotes the formation ofarteriosclerosis nidus through the migration of monocytes/macrophages(Current Therapy 12:1485-1488, 1994).

The relationship between tumor metastasis and adhesion moleculeabnormality has also been reported. For example, if E-cadherin-decreasedcancer cells showed strong invasiveness, but the invasiveness wasinhibited by introducing the cDNA of E-cadherin into the cancer cells,the invasiveness was recovered when E-cadherin antibodies antiserum wasadded to the cells. This suggests the tight relationship between thedecrease in the expression of E-cadherin and invasiveness of tumor cells(Frixen et al., 113:173, 1991). In actual clinical cases, therelationship between the decrease of the expression of E-cadherin andmetastasis is pointed out in various kinds of cancer such as hepatoma,esophageal cancer, gastric cancer, and breast cancer. It has also beenreported that “VLA-4” molecules, a ligand for “VCAM-1”, were highlyexpressed in metastatic melanoma, gastric cancer, and breast cancer,suggesting that this molecule can could be utilized for the implantationto vascular endothelial cells in metastasis. In addition, based onexperiments using various tumor cell lines, it has been reported thatepithelial cancer, such as gastric cancer, colon large intestine cancer,lung cancer, hepatoma, or pancreatic cancer, adhered to vascularendothelial cells through E-selectin (Takada et al., Cancer Res.,53:354, 1993).

On the other hand, therapeutic approach to treat diseases by targetingthese adhesion molecules have been made. For example, it was reportedthat anti-rat “ICAM-1” antibody strongly inhibited inflammatory reactionin rat autoimmune arthritis model. It has also been reported that theadministration of anti-“ICAM-1” antibody inhibited the onset ofarthritis in adjuvant synovitis in one of animal models of RA (Nihon etal., 14:571-577, 1991). It was further reported that the metastasisformation of inoculated tumor was remarkably inhibited if a large amountof peptides having REG sequence, which is that an amino acid sequence inan extracellular matrix protein recognized and bound by some integrins,were administered to a gallbladder cancer mouse, and that in in vitrosystem RGD peptides and anti-β1 subunit antibody inhibited the motionand infiltration of tumor cells (Yamada et al., Cancer Res., 50:4485,1990).

In the following, the present invention is described in detail byclarifying the meanings of terms used herein the present invention andthe general production methods of polypeptides, fusion polypeptides,genes, antibodies, transgenic mice, and knockout mice of the presentinvention. However, it is needless to say that the meanings of the termsare not to be interpreted limitedly by the definition given herein.

“Mitogen” used herein is also called also mitogenic factor and means asubstance which induces cell division. Immunologically, it means asubstance inducing blastogenesis of lymphocytes polyclonally andinducing cell division. Examples thereof are lectins such as PHA andPWM, Concanavalin A (ConA), lipopolysaccharides, streptolysin S, andanti-lymphocyte antibody. It is known that Concanavalin A and PHA actonly on T lymphocytes, that lipopolysaccharides act only on Blymphocytes, and that PWM acts on both lymphocytes.

The term “lymphoblast cell” used herein is also called also a largelymphocyte, lymphoblast, or immunoblast, and means a lymphocytebelonging to a large lymphocyte among lymphocytes existing in lymphoidtissues (lymph node, spleen, thymus, bone marrow, lymph duct, tonsil,etc.) and blood.

The term “activated lymphocyte” used herein, for example, a lymphocytementioned below, but is not limited thereto. For example, the term meansa lymphocyte activated by some stimulation. As mentioned above,lymphocytes are classified into T cells, B cells, and natural killercells. T cells are classified into CD4-positive cells and CD8-positivecells. Therefore, the “activated lymphocytes” of the present inventioninclude mainly activated T cells, activated B cells, and activatednatural killer cells, and activated T cells include activatedCD4-positive cells and activated CD8-positive cells.

Upon reacting with antigens presented by antigen-presenting cells,CD4-positive T cells secrete various cytokines, newly express receptorsfor these cytokines, enlarge their own size, start cell dividing,proliferate, and are activated. Activated CD4-positive T cells includethose in such a state.

CD8-positive T cells express IL-2R when they react with antigens. WhenIL-2 acts on IL-2R, the cells are differentiated into CTL, which hascellular cytotoxicity. CTL destroy their its target cells to kill themwhen they meet the same antigen peptide/MHC class I complex. WhenCD8-positive T cells are differentiated into CTL, granules increase inthe cytoplasm. These granules comprise various high molecular weightproteins, represented by perforin. Perforin resembles MAC composed ofthe fifth to ninth components of complement, and makes holes in the cellmembrane of target cells. The granules also comprise serine proteases,LT, and proteoglycan. If CD8-positive cells receive antigen stimulationand are differentiated into CTL, they also secrete lymphokines such asIFNγ, LT, TNF, or IL-2. Activated CD8-positive T cells include those insuch a state.

T cells show blast formation phenomenon when they react withhemagglutinin (phytohemagglutinin, PHA) or Concanavalin A (ConA).Activated T cells comprise include those in such a state.

B cells express B7 molecules, activate helper T cells by stimulatingCD28 on their surface with TCR, allow the helper T cells to expressCD40L or produce lymphokines. When the cells receive stimulation, theychange to expand their cell size or proliferate. Activated B cellsinclude those in such a state. In the present invention, activated Bcells include those secreting antibodies (antibody-secreting cells andplasma cells).

Activated natural killer cells mean those showing cytotoxic action ontumor cells or virus-infected cells as mentioned above. In the presentinvention, activated lymphocytes include thymus cells stimulated byConcanavalin A (ConA).

The “activated lymphoblast cell” used herein includes an activated“lymphoblast” that is generated when the lymphoblast mentioned above isstimulated with “mitogen” mentioned above such as Concanavalin A.

The term “resting lymphocyte” used herein, in some case, annon-activated lymphocyte, which has not received the stimulation toactivate cells, in contrast to an activated lymphocyte mentioned above.

The “gene” of the present invention includes a genomic DNA and a cDNA.

The “human-derived” substance of the present invention includes naturalsubstance isolated from a human body component (organ, tissue, cell,body fluid, etc.), and recombinant substance produced by recombinant DNAtechnology. When the substance is protein or polypeptide, the substanceincludes an artificial protein and polypeptide having an amino acidsequence where one or more amino acids are substituted, deleted, oradded.

The “cell surface molecule” of the present invention is that derivedfrom a mammal such as human, rat, mouse, guinea pig, and rabbit,preferably that derived from human, rat, or mouse, and more preferablythat derived from human.

Specifically, the “cell surface molecule” of the present invention isthat characterized by having, at least, properties described below:

(a) the cell surface molecule is expressed in, at least, thymocytes andmitogen-stimulated lymphoblast cells;

(b) an antibody reactive to the cell surface molecule induces adhesionbetween mitogen-stimulated lymphoblast cells;

(c) an antibody reactive to the cell surface molecule inducesproliferation of peripheral blood lymphocytes under the coexistencewithin the presence of an antibody against CD3;

(d) the cell surface molecule has a partial amino acid sequencerepresented by Phe-Asp-Pro-Pro-Pro-Phe (SEQ ID NO:21) in itsextracellular region; and

(e) the cell surface molecule has a partial amino acid sequencerepresented by Tyr-Met-Phe-Met (SEQ ID NO:22) in its cytoplasmic region.

Preferably, the “cell surface molecule” comprises the following“polypeptide” of the present invention.

The “polypeptide” of the present invention is that which constitutes theabove-mentioned “cell surface molecule” of the present invention.Examples thereof are as follows.

(1) A polypeptide encoded by a DNA hybridizing with a DNA comprising anucleotide sequence of SEQ ID NO: 1 under stringent conditions;

(2) A polypeptide having an amino acid sequence having 60% or morehomology with an amino acid sequence of SEQ ID NO: 2;

(3) A polypeptide having an amino acid sequence of SEQ ID NO: 2 or anamino acid sequence substantially the same as the amino acid sequence(namely, a polypeptide constituting “human JTT-1 antigen” and itsderivative);

(4) A polypeptide having an amino acid sequence encoded by a nucleotidesequence corresponding to nucleotide residues 26 to 625 of SEQ ID NO: 3or an amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting “human JTT-1 antigen” andits derivative);

(5) A polypeptide having an amino acid sequence encoded by a nucleotidesequence corresponding to nucleotide residues 35 to 637 of SEQ ID NO: 4or an amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting “rat JTT-1 antigen” and itsderivative);

(6) A polypeptide having an amino acid sequence encoded by a nucleotidesequence corresponding to nucleotide residues 1 to 603 of SEQ ID NO: 5or an amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting “mouse JTT-1 antigen” andits derivative);

(7) A polypeptide having an amino acid sequence encoded by a nucleotidesequence corresponding to nucleotide residues 35 to 685 of SEQ ID NO: 6or an amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting a “mutant of rat JTT-1antigen” and its derivative); and

(8) A polypeptide having an amino acid sequence encoded by a DNAencoding a polypeptide constituting the cell surface molecule of thepresent invention, wherein the DNA is introduced into the transformantidentified by an international deposit accession No. FERM BP-5725 or,having amino acid sequence substantially the same as the amino acidsequence (namely, a polypeptide constituting a “human JTT-1 antigen” andits derivative).

To determine the “percent homology” of two amino acid sequences or oftwo nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlapping positions)×100). In one embodiment the two sequences are the same length.

To determine percent homology between two sequences, the algorithm ofKarlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268,modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-5877 is used. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul, et al. (1990) J. Mol. Biol.215:403-410. BLAST nucleotide searches are performed with the NBLASTprogram, score=100, word length=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules of the invention. BLAST proteinsearches are performed with the XBLAST program, score=50, word length=3to obtain amino acid sequences homologous to a VRK1 or VRK2 proteinmolecules. To obtain gapped alignments for comparison purposes, GappedBLAST is utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used. See web site of National Center for Biotechnology Information(NCBI), which is a division of National Library of Medicine (NLM) at theNational Institutes of Health of USA.

Furthermore, the present invention relates to a DNA that specificallyhybridizes under moderate or highly stringent conditions to a DNAencoding a protein of the present invention and comprises at least 15nucleotide residues. The DNA can be used, for example, as a probe todetect or isolate a DNA encoding a protein of the present invention, oras a primer for PCR amplification. An example is DNA consisting of atleast 15 nucleotides complementary to the nucleotide sequence of SEQ IDNO: 1, NO: 3, NO:4, NO:5 or NO:6.

Standard hybridization conditions (e.g., moderate or highly stringentconditions) are known to those skilled in the art and can be found inCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6, hereby incorporated by reference. Moderate hybridizationconditions are defined as equivalent to hybridization in 2× sodiumchloride/sodium citrate (SSC) at 30° C., followed by one or more washesin 1×SSC, 0.1% SDS at 50-60° C. Highly stringent conditions are definedas equivalent to hybridization in 6× sodium chloride/sodium citrate(SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at50-65° C.

Examples of “stringent conditions” are as follows. When a probe with 50or more nucleotides is used and hybridization is performed in 0.9% NaCl,the standard of temperature where 50% dissociation occurs (Tm) iscalculated using the following formula and the temperature forhybridization can be determined according to the following formula.Tm=82.3° C.+0.41×(G+C) % −500/n−0.61×(formamide) % (n means the numberof the nucleotide of probe).Temperature=Tm−25° C.

In addition, when a probe with 100 or more nucleotides (G+C=40 to −50%)is used, it should be considered that Tm varies as (1) and (2) mentionedbelow.

(1) Tm descends by about 1° C. per 1% mismatch.

(2) Tm descends by 0.6 to 0.7° C. per 1% formamide.

Accordingly, the temperature conditions for the combination ofcompletely complementary strands can be set as follows.

(A) 65 to 75° C. (formamide not added)

(B) 35 to 45° C. (in the presence of 50% formamide)

The temperature conditions for the combination of incompletelycomplementary strands can be set as follows.

(A) 45 to 55° C. (formamide not added

(B) 35 to 42° C. (in the presence of 30% formamide)

The temperature conditions when a probe with 23 or less nucleotides isused can be 37° C. or can be calculated using the following formula.Temperature=2° C.×(the number of A+T)+4° C.×(the number of C+G)−5° C.

Here, “having substantially the same amino acid sequence” means toinclude a polypeptide having an amino acid sequence where multiple aminoacids, preferably 1 to 10 amino acids, particularly preferably 1 to 5amino acids, in the amino acid sequence shown in Sequence Listing aresubstituted, deleted, and/or modified, and a polypeptide having an aminoacid sequence where multiple amino acids, preferably 1 to 10 aminoacids, particularly preferably 1 to 5 amino acids, are added to theamino acid sequence shown in Sequence Listing, as long as thepolypeptide has substantially the same biological properties as thepolypeptide having the amino acid sequence shown in Sequence Listing.

Such substitution, deletion, or insertion of amino acids can beperformed by the usual method (Experimental Medicine: SUPPLEMENT,“Handbook of Genetic Engineering” (1992); and so on).

Examples thereof are synthetic oligonucleotide-directed mutagenesis(gapped duplex method), point metagenesis by which a point mutation isintroduced at random by treatment with nitrite or sulfite, the method bywhich a deletion mutant is prepared with Bal3l enzyme and the like,cassette mutagenesis, linker scanning method, miss incorporation method,mismatch primer method, DNA segment synthesis method, etc.

Synthetic oligonucleotide-directed mutagenesis (gapped duplex method)can be, for example, performed as follows. The region desired to bemutagenized is cloned into M13 phage vector having amber mutation toprepare the single-stranded phage DNA. After RF I DNA of M13 vectorwithout amber mutation is linearized by restriction enzyme treatment,DNA is mixed with the single-stranded phage DNA mentioned above,denatured, and annealed thereby forming “gapped duplex DNA.” A syntheticoligonucleotide into which mutations are introduced is hybridized withthe gapped duplex DNA and the closed-circular double-stranded DNAs areprepared by the reactions with DNA polymerase and DNA ligase. E. colimutS cells, deficient in mismatch repair activity, are transfected withthis DNA., E. coli cells without suppressor activity are infected withthe grown phages, and only phages without amber mutation are screened.

The method by which a point mutation is introduced with nitriteutilizes, for example, the principle as mentioned below. If DNA istreated with nitrite, bases are deaminated to change adenine intohypoxanthine, cytosine into uracil, and guanine into xanthine. Ifdeaminated DNA is introduced into cells, “A:T” and “G:C” are replacedwith “G:C” and “A:T”, respectively, because hypoxanthine, uracil, andxanthine form a base pair with cytosine, adenine, and thymine,respectively, in the DNA replication. Actually, single-stranded DNAfragments treated with nitrite are hybridized with “gapped duplex DNA”,and thereafter mutant strains are separated by manipulating in the sameway as synthetic oligonucleotide-directed mutagenesis (gapped duplexmethod).

Conservative amino acid substitutions can also be made at one or morepredicted non-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

Alphabetical triplet or single letter codes used to represent aminoacids in the present specification or figures mean amino acids asfollows. (Gly/G) glycine, (Ala/A) alanine, (Val/V) valine, (Leu/L)leucine, (Ile/I) isoleucine, (Ser/S) serine, (Thr/T) threonine, (Asp/D)aspartic acid, (Glu/E) glutamic acid, (Asn/N) asparagine, (Gln/Q)glutamine, (Lys/K) lysine, (Arg/R) arginine, (Cys/C) cysteine, (Met/M)methionine, (Phe/F) phenylalanine, (Tyr/Y) tyrosine, (Trp/W)tryptophane, (His/H) histidine, (Pro/P) proline.

The “polypeptide” constituting the above-mentioned “cell surfacemolecule” of the present invention is a transmembrane protein, whichpenetrates cell membrane, and the “cell surface molecule” is composed ofone or two of these transmembrane polypeptides.

Here, a “transmembrane protein” means a protein that connects withmembrane through the hydrophobic peptide region penetrating the lipidbilayer of the membrane once or several times and whose structure is, asa whole, composed of three main regions, that is, extracellular region,transmembrane region, and cytoplasmic region, as seen in many receptorsor cell surface molecules. Such a transmembrane protein constitutes eachreceptor or cell surface molecule by existing in the form of a monomer,homodimer, heterodimer or oligomer with another chain(s) having the sameor different amino acid sequence.

The “polypeptide fragment” of the present invention is a fragment fromthe above-defined “polypeptide” of the present invention, and preferablythe extracellular region of the polypeptide. One to five amino acids, ifdesired, can be added to the N terminus and/or C terminus of thisregion.

Here, an “extracellular region” means the whole or a portion from thepartial structure (partial region) from the entire structure of theabove-mentioned transmembrane protein where the partial structure existsoutside of the membrane. In other words, it means the whole or a portionof the region of the transmembrane protein except the region integratedincorporated into the membrane (transmembrane region) and the regionexisting in the cytoplasm following the transmembrane region in themembrane (cytoplasmic regions).

“The constant region or a portion of the constant region of humanimmunoglobulin (Ig) heavy chain” used herein means the constant regionor the Fc region of human-derived immunoglobulin heavy chain (H chain)as described above, or a portion of them. The immunoglobulin can be anyimmunoglobulin belonging to any class and any subclass. Specifically,examples of the immunoglobulin are IgG (IgG1, IgG2, IgG3, and IgG4),IgM, IgA (IgA1 and IgA2), IgD, and IgE. Preferably, the immunoglobulinis IgG (IgG1, IgG2, IgG3, or IgG4), or IgM. Examples of particularlypreferable immunoglobulin in of the present invention are thosebelonging to human-derived IgG (IgG1, IgG2, IgG3, or IgG4).

Immunoglobulin has a Y-shaped structural unit in which four chainscomposed of two homologous light chains (L chains) and two homologousheavy chains (H chains) are connected through disulfide bonds (S-Sbonds). The light chain is composed of the light chain variable regions(VL) and the light chain constant region (CL). The heavy chain iscomposed of the heavy chain variable regions (VH) and the heavy chainconstant region (CH).

The heavy chain constant region is composed of some domains having theamino acid sequences inherent in each class (IgG, IgM, IgA, IgD, andIgE) and each subclass (IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2).

The heavy chain of IgG (IgG1, IgG2, IgG3, and IgG4) is composed of VH,CH1 domain, hinge region, CH2 domain, and CH3 domain in this order fromN terminus.

Similarly, the heavy chain of IgG1 is composed of VH, Cγ₁1 domain, hingeregion, Cγ₁2 domain, and Cγ₁3 domain in this order from N terminus. Theheavy chain of IgG2 is composed of VH, Cγ₂1 domain, hinge region, Cγ₂2domain, and Cγ₂3 domain in this order from N terminus. The heavy chainof IgG3 is composed of VH, Cγ₃1 domain, hinge region, Cγ₃ ² domain, andCγ₃3 domain in this order from N terminus. The heavy chain of IgG4 iscomposed of VH, Cγ₄1 domain, hinge region, Cγ₄2 domain, and Cγ₄3 domainin this order from N terminus.

The heavy chain of IgA is composed of VH, Cα1 domain, hinge region, Cα2domain, and Cα3 domain in this order from N terminus.

Similarly, the heavy chain of IgA1 is composed of VH, Cα₁1 domain, hingeregion, Cα₁2 domain, and Cα₁3 domain in this order from N terminus. Theheavy chain of IgA2 is composed of VH, Cα₂1 domain, hinge region, Cα₂2domain, and Cα₂3 domain in this order from N terminus.

The heavy chain of IgD is composed of VH, Cδ1 domain, hinge region, Cδ2domain, and Cδ3 domain in this order from N terminus.

The heavy chain of IgM is composed of VH, Cμ1 domain, Cμ2 domain, Cμ3domain, and Cε4 domain in this order from N terminus and have no hingeregion as seen in IgG, IgA, and IgD.

The heavy chain of IgE is composed of VH, Cε1 domain, Cε2 domain, Cε3domain, and Cε4 domain in this order from N terminus and have no hingeregion as seen in IgG, IgA, and IgD.

If, for example, IgG is treated with papain, it is cleaved at theslightly N terminal side beyond the disulfide bonds existing in thehinge region where the disulfide bonds connect the two heavy chains togenerate two homologous Fab, in which a heavy chain fragment composed ofVH and CH1 is connected with one light chain through a disulfide bond,and one Fc, in which two homologous heavy chain fragments composed ofthe hinge region, CH2 domain, and CH3 domain are connected throughdisulfide bonds (See “Immunology Illustrated”, original 2nd ed.,Nankodo, pp. 65-75 (1992); and “Focus of Newest Medical Science‘Recognition Mechanism of Immune System’”, Nankodo, pp. 4-7 (1991); andso on).

Namely, “a portion of a constant region of immunoglobulin heavy chain”of the present invention means a portion of a constant region of animmunoglobulin heavy chain having the structural characteristics asmentioned above, and preferably, is the constant region without C1domain, or the Fc region. Specifically, examples thereof are the regioncomposed of hinge region, C2 domain, and C3 domain in the case from eachof IgG, IgA, and IgD, and are the region composed of C2 domain, C3domain, and C4 domain in the case from each of IgM and IgE. Aparticularly preferable example thereof is the Fc region ofhuman-derived IgG1.

The “fusion polypeptide” of the present invention is that composed ofthe extracellular region of the “polypeptide” constituting theabove-described “cell surface molecule” of the present invention and “aconstant region or a portion of a constant region of humanimmunoglobulin (Ig) heavy chain.” Preferably, it is a fusion polypeptidecomposed of an extracellular region of a polypeptide of the presentinvention and a portion of a constant region of human IgG heavy chain,and particularly preferably, it is a fusion polypeptide composed of anextracellular region of a polypeptide of the present invention and theregion (Fc) composed of a hinge region, CH2 domain, and CH3 domain ofhuman IgG heavy chain. Moreover, IgG1 is preferable among IgG. Inaddition, a polypeptide derived from human, mouse, or rat (preferably,human) is preferable as the polypeptide of the present invention.

The fusion polypeptide of the present invention has the advantage thatthe fusion polypeptide can be purified extremely easily by usingaffinity column chromatography using the property of protein A, whichbinds specifically to the immunoglobulin fragment because the fusionpolypeptide of the present invention has a portion of a constant region(for example Fc) of an immunoglobulin such as IgG as mentioned above asa fusion partner. Moreover, since various antibodies against the Fc ofvarious immunoglobulin are available, an immunoassay for the fusionpolypeptides can be easily performed with antibodies against the Fc.

The polypeptide, polypeptide fragment, and fusion polypeptide of thepresent invention can be produced not only by recombinant DNA technologyas mentioned below but also by a method well known in the art such as achemical synthetic method and a cell culture method, or a modifiedmethod thereof.

The “gene” of the present invention comprises a DNA encoding theabove-mentioned polypeptide or polypeptide fragment of the presentinvention, and includes any gene having a nucleotide sequence encodingthe polypeptide or polypeptide fragment of the present invention.

Examples of the gene are those encoding the polypeptide or polypeptidefragment mentioned below.

(1) A polypeptide encoded by a DNA hybridizing with a DNA comprising anucleotide sequence of SEQ ID NO: 1 under stringent conditions;

(2) A polypeptide having an amino acid sequence having 60% or morehomology with an amino acid sequence of SEQ ID NO: 2;

(3) A polypeptide having an amino acid sequence of SEQ ID NO: 2 or anamino acid sequence substantially the same as the amino acid sequence(namely, a polypeptide constituting “human JTT-1 antigen” and itsderivative);

(4) A polypeptide having an amino acid sequence encoded by a nucleotidesequence corresponding to nucleotide residues 26-625 of SEQ ID NO: 3 oran amino acid sequence substantially the same as the amino acid sequence(namely, a polypeptide constituting “human JTT-1 antigen” and itsderivative);

(5) A polypeptide having an amino acid sequence encoded by a nucleotidesequence corresponding to nucleotide residues 35-637 of SEQ ID NO: 4 oran amino acid sequence substantially the same as the amino acid sequence(namely, a polypeptide constituting “rat JTT-1 antigen” and itsderivative);

(6) A polypeptide having an amino acid sequence encoded by a nucleotidesequence corresponding to nucleotide residues 1-603 of SEQ ID NO: 5 oran amino acid sequence substantially the same as the amino acid sequence(namely, a polypeptide constituting “mouse JTT-1 antigen” and itsderivative);

(7) A polypeptide having an amino acid sequence encoded by a nucleotidesequence corresponding to nucleotide residues 35-685 of SEQ ID NO: 6 oran amino acid sequence substantially the same as the amino acid sequence(namely, a polypeptide constituting a “mutant of rat JTT-1 antigen” andits derivative); and

(8) A polypeptide having an amino acid sequence encoded by a DNAencoding a polypeptide constituting the cell surface molecule of thepresent invention, wherein the DNA is introduced into the transformantidentified by an international deposit accession No. FERM BP-5725 or,having an amino acid sequence substantially the same as said amino acidsequence (namely, a polypeptide constituting a “human JTT-1 antigen” andits derivative).

Here, “substantially the same amino acid sequence” means as definedabove.

Specific examples of the gene of the present invention are DNAs or theirfragments mentioned below.

(1) A DNA comprising a nucleotide sequence of SEQ ID NO: 1, and a DNAhybridizing with the DNA under stringent conditions;

(4) A DNA comprising a nucleotide sequence corresponding to nucleotideresidues 26-625 of SEQ ID NO: 3;

(5) A DNA comprising a nucleotide sequence corresponding to nucleotideresidues 35-637 of SEQ ID NO: 4;

(6) A DNA comprising a nucleotide sequence corresponding to nucleotideresidues 1-603 of SEQ ID NO: 5;

(7) A DNA comprising a nucleotide sequence corresponding to nucleotideresidues 35-685 of SEQ ID NO: 6;

(8) A DNA encoding a polypeptide constituting a cell surface molecule ofthe present invention, wherein the DNA is introduced into a transformantidentified by an international deposit accession No. FERM BP-5725.

The DNA encoding a portion of a constant region of immunoglobulin heavychain, which is a part of a fusion polypeptide of the present invention,can be cDNA, or genomic DNA comprised of intons between every exon (theDNA encoding, for example, CH1 domain, hinge region, CH2 domain, CH3domain, CH4 domain and so on).

The DNA of the present invention includes any DNA comprised of anycodons as long as the codons encode the same amino acids.

The DNA of the present invention can be a DNA obtained by any method.For example, the DNA includes complementary DNA (cDNA) prepared frommRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis,DNA obtained by PCR amplification with RNA or DNA as a template, and DNAconstructed by appropriately combining these methods.

The DNA encoding the polypeptide of the present invention can beobtained by the usual method such as a method to clone cDNA from mRNAencoding the polypeptide of the present invention, a method to isolategenomic DNA and then splice them, chemical synthesis and so on.

(1) cDNA can be cloned from the mRNA encoding the polypeptide of thepresent invention by, for example, the method described below.

First, the mRNA encoding a cell surface molecule (polypeptide) of thepresent invention is prepared from tissues or cells (for example, thymuscells or spleen-derived lymphoblast cells stimulated with ConA)expressing and producing a cell surface molecule (polypeptide) of thepresent invention. mRNA can be prepared isolating total RNA by a knownmethod such as quanidine-thiocyanate method (Chirgwin et al.,Biochemistry, 18:5294, 1979), hot phenol method, or AGPC method, andsubjecting it to affinity chromatography using oligo-dT cellulose orpoly-U Sepharose.

Then, with the mRNA obtained as a template, cDNA is synthesized, forexample, by a well-known method using reverse transcriptase such as themethod of Okayama et al. (Mol. Cell. Biol. 2:161, 1982; ibid. 3:280,1983) or the method of Hoffman et al. (Gene 25:263, 1983), and convertedinto double-stranded cDNA. A cDNA library is prepared by transforming E.coli with plasmid vectors, phage vectors, or cosmid vectors having thiscDNA or by transfecting E. coli after in vitro packaging.

The plasmid vectors used in this invention are not limited as long asthey are replicated and maintained in hosts. Any phage vectors that canbe replicated in hosts can also be used. Examples of usually usedcloning vectors are pME18S, λZAPII(lZAPII), pUC19, λgt10, λgt11, and soon. When the vector is applied to immunological screening as mentionedbelow, the vector having a promoter that can express a gene encoding thepolypeptide of the present invention in a host is preferably used.

cDNA can be inserted into a plasmid by, for example, the method ofManiatis et al. (Molecular Cloning, A Laboratory Manual, second edition,Cold Spring Harbor Laboratory, p. 1.53, 1989). cDNA can be inserted intoa phage vector by, for example, the method of Hyunh et al. (DNA cloning,a practical approach, Vol. 1, p. 49 (1985)). These methods can be simplyperformed by using a commercially available cloning kit (for example, aproduct from Takara Shuzo). The recombinant plasmid or phage vector thusobtained is introduced into appropriate host cells such as a prokaryote(for example, E. coli: XL1Blue MRF′, DH5α, HB101, MC1061/P3, etc.).

Examples of a method for introducing a plasmid into a host are calciumchloride method, calcium chloride/rubidium chloride method described inMolecular Cloning, A Laboratory Manual (second edition, Cold SpringHarbor Laboratory, p. 1.74 (1989)), and electroporation method. Phagevectors can be introduced into host cells by, for example, a method inwhich the phage DNAs are introduced into grown hosts after in vitropackaging. In vitro packaging can be easily performed with acommercially available in vitro packaging kit (for example, a productfrom Stratagene or Amersham).

The cDNA encoding the polypeptide of the present invention can beisolated from the cDNA library so prepared according to the methodmentioned above by combining general cDNA screening methods.

For example, a clone comprising the desired cDNA can be screened by aknown colony hybridization method (Crunstein et al., Proc. Natl. Acad.Sci. USA, 72:3961, 1975) or plaque hybridization method (MolecularCloning, A Laboratory Manual, second edition, Cold Spring HarborLaboratory, p. 2.108 (1989)) using ³²P-labeled chemically synthesizedoligonucleotides as probes, which are corresponding to the amino acidsequence of the polypeptide of the present invention. Alternatively, aclone having a DNA fragment encoding a specific region within thepolypeptide of the present invention can be screened by amplifying theregion by PCR with synthetic PCR primers.

When a cDNA library prepared using a cDNA expression vector (forexample, λZAPII phage vector) is used, the desired clone can be screenedby the antigen-antibody reaction using an antibody against thepolypeptide of the present invention. A screening method using PCRmethod is preferably used when many clones are subjected to screening.

The nucleotide sequence of the DNA thus obtained can be determined byMaxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA, 74:560,1977) or the dideoxynucleotide synthetic chain termination method usingphage M13 (Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467,1977). The whole or a portion of the gene encoding the polypeptide ofthe present invention can be obtained by excising the clone obtained asmentioned above with restriction enzymes and so on.

(2) The DNA encoding the polypeptide of the present invention can beisolated from the genomic DNA derived from the cells expressing thepolypeptide of the present invention as mentioned above by the followingmethods.

Such cells are solubilized preferably by SDS or proteinase K, and theDNAs are deproteinized by repeating phenol extraction. RNAs are digestedpreferably with ribonuclease. The DNAs obtained are partially digestedwith appropriate restriction enzymes, and the DNA fragments obtained areamplified with appropriate phage or cosmid to generate a library. Then,clones having the desired sequence are detected, for example, by usingradioactively labeled DNA probes, and the whole or a portion of the geneencoding the polypeptide of the present invention is obtained from theclones by excision with restriction enzyme and so on.

cDNA encoding a human-derived polypeptide can be obtained as follows.After a cosmid library into which human genomic DNA (chromosomal DNA) isintroduced is prepared (“Laboratory Manual: Human Genome Mapping”,Maruzen press), positive clones comprising the DNA of the coding regionof the desired protein are obtained by screening the cosmid library.Then, the cDNA library mentioned above is screened with the coding DNAexcised from the positive clone as a probe to prepare the human cDNA.

(3) The DNA of the present invention can also be chemically synthesizedby the usual method, based on the nucleotide sequence of SEQ ID NO: 1,3, 4, 5, or 6.

The present invention also relates to a recombinant vector comprisingthe DNA encoding an above-mentioned cell surface molecule (polypeptide)of the present invention. The recombinant vector of the presentinvention is not limited as long as it can be replicated and maintainedor can autonomously replicate in various prokaryotic and/or eukaryotichosts. The vector of the present invention includes plasmid vectors andphage vectors.

The recombinant vector can easily be prepared by ligating the DNAencoding the polypeptide of the present invention with a vector forrecombination available in the art (plasmid DNA and bacteriophage DNA)by the usual method. Specific examples of the vectors for recombinationused are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13,and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillussubtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples ofphages are a bacteriophage such as λ phage, and an animal or insectvirus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, andnuclear polyhidrosis virus.

An expression vector is useful for expressing the DNA encoding thepolypeptide of the present invention and for producing the polypeptideof the present invention. The expression vector is not limited as longas it expresses the gene encoding the polypeptide of the presentinvention in various prokaryotic and/or eukaryotic host cells andproduces this protein. Examples thereof are pEFneo (Proc. Natl. Acad.Sci. USA 91:158-162, 1994), pEF-BOS (Nucleic Acids Res. 18:5322, 1990),pME18S (Experimental Medicine: SUPPLEMENT, “Handbook of GeneticEngineering” (1992)), pMAL C2, and so on.

When bacteria, particularly E. coli are used as host cells, anexpression vector is generally comprised of, at least, apromoter/operator region, an initiation codon, the DNA encoding thepolypeptide of the present invention, termination codon, terminatorregion, and replicon.

When yeast, animal cells, or insect cells are used as hosts, anexpression vector is preferably comprised of, at least, a promoter, aninitiation codon, the DNA encoding the polypeptide of the presentinvention, and a termination codon. It may also comprise the DNAencoding a signal peptide, enhancer sequence, 5′- and 3′-untranslatedregion of the gene encoding the polypeptide of the present invention,splicing junctions, polyadenylation site, selectable marker region, andreplicon. The expression vector may also contain, if required, a genefor gene amplification (marker) that is usually used.

A promoter/operator region to express the polypeptide of the presentinvention in bacteria comprises a promoter, an operator, and aShine-Dalgarno (SD) sequence (for example, AAGG). For example, when thehost is Escherichia, it preferably comprises Trp promoter, lac promoter,recA promoter, λPL promoter, lpp promoter, tac promoter, or the like.Examples of a promoter to express the polypeptide of the presentinvention in yeast are PH05 promoter, PGK promoter, GAP promoter, ADHpromoter, and so on. When the host is Bacillus, examples thereof areSL01 promoter, SP02 promoter, penP promoter and so on. When the host isa eukaryotic cell such as a mammalian cell, examples thereof areSV40-derived promoter, retrovirus promoter, heat shock promoter, EFpromoter, and so on, and preferably SV-40, SRα, and retrovirus-derivedone. As a matter of course, the promoter is not limited to the aboveexamples. In addition, to use an enhancer is effective for expression.

A preferable initiation codon is, for example, a methionine codon (ATG).

The commonly used termination codon (for example, TAG, TGA, TAA, and soon) is illustrated as a termination codon.

Usually used natural or synthetic terminators are used as a terminatorregion.

A replicon means a DNA capable of replicating the whole DNA sequence inhost cells, and includes a natural plasmid, an artificially modifiedplasmid (DNA fragment prepared from a natural plasmid), a syntheticplasmid, and so on. Examples of a preferable plasmids are pBR322 or itsartificial derivatives (DNA fragment obtained by treating pBR322 withappropriate restriction enzymes) for E. coli, yeast 2μ plasmid or yeastchromosomal DNA for yeast, and pEFneo, pME18S, pRSVneo ATCC 37198,pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, etc.,for mammalian cells.

An enhancer sequence, polyadenylation site, and splicing junction thatare usually used in the art, such as those derived from SV40 can be alsoused.

A selectable marker usually used can be used according to the usualmethod. Examples thereof are resistance genes for antibiotics, such astetracycline, neomycin, ampicillin, or kanamycin, and thymidine kinasegene.

Examples of a gene for gene amplification are dihydrofolate reductase(DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamatesynthase gene, adenosine deaminase gene, ornithine decarboxylase gene,hygromycin-B-phophotransferase gene, aspartate transcarbamylase gene,etc.

The expression vector of the present invention can be prepared bycontinuously and circularly linking at least the above-mentionedpromoter, initiation codon, DNA (gene) encoding the polypeptide of thepresent invention, termination codon, and terminator region, to anappropriate replicon. If desired, appropriate DNA fragments (forexample, linkers, restriction sites generated with other restrictionenzyme), can be used by the usual method such as digestion with arestriction enzyme or ligation using T4 DNA ligase.

Transformants of the present invention can be prepared by introducingthe expression vector mentioned above into host cells.

Host cells used in the present invention are not limited as long as theyare compatible with an expression vector mentioned above and can betransformed. Examples thereof are various cells such as natural cells orartificially established recombinant cells usually used in technicalfield of the present invention (for example, bacteria (Escherichia andBacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, or insectcells.

E. coli or animal cells are preferably used. Specific examples are E.coli (DH5α, XL1Blue MRF′, TB1, HB101, etc.), mouse-derived cells (COP,L, C127, Sp2/0,NS-1, NIH 3T3, etc.), rat-derived cells, hamster-derivedcells (BHK, CHO-K1, CHO, etc.), monkey-derived cells (COS1, COS3, COS7,CV1, Velo, etc.), and human-derived cells (HEK293, Hela, diploidfibroblast-derived cells, myeloma, Namalwa, etc.).

An expression vector can be introduced (transformed (transduced)) intohost cells by known method.

Transformation can be performed, for example, according to the method ofCohen et al. (Proc. Natl. Acad. Sci. USA 69:2110, 1972), protoplastmethod (Mol. Gen. Genet. 168:111, 1979), or competent method (J. Mol.Biol. 56:209, 1971) when the hosts are bacteria (E. coli, Bacillussubtilis, etc.), the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA75:1927, 1978), or lithium method (J. Bacteriol. 153:163, 1983) when thehost is Saccharomyces cerevisiae, the method of Graham (Virology 52:456,1973) when the hosts are animal cells, and the method of Summers et al.(Mol. Cell. Biol. 3:2156-2165, 1983) when the hosts are insect cells.

The polypeptide of the present invention can be produced by cultivatingtransformants (in the following this term includes transductants)comprising an expression vector prepared as mentioned above in nutrientmedia.

The nutrient media preferably comprise carbon source, inorganic nitrogensource, or organic nitrogen source necessary for the growth of hostcells (transformants). Examples of the carbon source are glucose,dextran, soluble starch, and sucrose, and examples of the inorganic ororganic nitrogen source are ammonium salts, nitrates, amino acids, cornsteep liquor, peptone, casein, meet extract, soy bean cake, and potatoextract. If desired, they may comprise other nutrients (for example, aninorganic salt (for example, calcium chloride, sodiumdihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (forexample, tetracycline, neomycin, ampicillin, kanamycin, etc.).

Cultivation is performed by a method known in the art. Cultivationconditions such as temperature, pH of the media, and cultivation timeare selected appropriately so that the polypeptide of the presentinvention is overproduced.

Specific media and cultivation conditions used depending on host cellsare illustrated below, but are not limited thereto.

When the hosts are bacteria, actinomycetes, yeasts, filamentous fungi,liquid media comprising the nutrient source mentioned above areappropriate. The media with pH 5 to 8 are preferably used.

When the host is E. coli, examples of preferable media are LB media, andM9 media (Miller et al., Exp. Mol. Genet., Cold Spring HarborLaboratory, p. 431 (1972)). Using these media, cultivation can beperformed usually at 14 to 43° C. for about 3 to 24 hours with aerationand stirring, if necessary.

When the host is Bacillus, cultivation can be performed usually at 30 to40° C. for about 16 to 96 hours with aeration and stirring, ifnecessary.

When the host is yeast, examples of media are Burkholder minimal media(Bostian, Proc. Natl. Acad. Sci. USA, 77:4505, 1980). The pH of themedia is preferably 5 to 8. Cultivation can be performed usually at 20to 35° C. for about 14 to 144 hours with aeration and stirring, ifnecessary.

When the host is an animal cell, examples of media are MEM mediacontaining about 5 to 20% fetal bovine serum (Science 122:501, 1952),DMEM media (Virology 8:396, 1959), RPMI1640 media (J. Am. Med. Assoc.199:519, 1967), and 199 media (Proc. Soc. Exp. Biol. Med. 73:1, 1950).The pH of the media is preferably about 6 to 8. Cultivation can beperformed usually at about 30 to 40° C. for about 15 to 72 hours withaeration and stirring, if necessary.

When the host is an insect cell, an example of media is Grace's mediacontaining fetal bovine serum (Proc. Natl. Acad. Sci. USA 82:8404,1985). The pH thereof is preferably about 5 to 8. Cultivation can beperformed usually at about 20 to 40° C. for 15 to 100 hours withaeration and stirring, if necessary.

Cultivation of transformants as mentioned above, in particular animalcells can overexpress the polypeptide of the present invention on thesurface of the cells.

The polypeptide of the present invention can be produced as a solublepolypeptide fragment such as an extracellular region fragment bypreparing the transformants as mentioned above using the DNA encodingthe extracellular region or each domain and by cultivating thetransformants to allow them to secrete the soluble polypeptide into theculture supernatant. In addition, a fusion polypeptide of the presentinvention can be prepared similarly.

Namely, a culture filtrate (supernatant) is obtained by the method suchas filtration or centrifugation of the obtained culture, and thepolypeptide or polypeptide fragment of the present invention is purifiedand isolated from the culture filtrate by the usual method commonly usedin order to purify and isolate a natural or synthetic protein.

Examples of the isolation and purification method are a method utilizingsolubility, such as salting out and solvent precipitation method, amethod utilizing the difference in molecular weight, such as dialysis,ultrafiltration, gel filtration, and sodium dodecylsulfate-polyacrylamide gel electrophoresis, a method utilizing charges,such as ion exchange chromatography and hydroxylapatite chromatography,a method utilizing specific affinity, such as affinity chromatography, amethod utilizing the difference in hydrophobicity, such as reverse phasehigh performance liquid chromatography, and a method utilizing thedifference in isoelectric point, such as isoelectric focusing.

When the polypeptide or a polypeptide fragment of the present inventionexists in the periplasm or cytoplasm of cultured transformants, first,the fungus bodies or cells are harvested by the usual method such asfiltration or centrifugation and suspended in appropriate buffer. Afterthe cell wall and/or cell membrane of the cells and so on are disruptedby the method such as lysis with sonication, lysozyme, andfreeze-thawing, the membrane fraction comprising the polypeptide of thepresent invention is obtained by the method such as centrifugation orfiltration. The membrane fraction is solubilized with a detergent suchas Triton-X100 to obtain the crude extract. Finally, the polypeptide orthe polypeptide fragment is isolated and purified from the crude extractby the usual method as illustrated above.

The “transgenic mouse” of the present invention is a transgenic mousewherein the DNA (cDNA or genomic DNA) prepared as mentioned aboveencoding the polypeptide of the present invention derived from animalsexcept mice (non-self polypeptide) have been integrated into itsendogenous locus of the mouse. The transgenic mouse expresses thenon-self polypeptide and secretes the polypeptide into its body.

The transgenic mouse can be prepared according to the method as usuallyused for producing a transgenic animal (for example, see “Newest Manualof Animal Cell Experiment”, LIC press, Chapter 7, pp. 361-408, (1990)).

Specifically, for example, embryonic stem cells (ES cells) obtained fromnormal mouse blastocysts are transformed with an expression vector inwhich the gene encoding human-derived polypeptide of the presentinvention (i.e., “human JTT-1 antigen”) has been operably inserted. EScells in which the gene encoding the human-derived polypeptide of thepresent invention has been integrated into the endogenous gene arescreened by the usual method. Then, the ES cells screened aremicroinjected into a fertilized egg obtained from another normal mouse(blastocyst) (Proc. Natl. Acad. Sci. USA 77:7380-7384, 1980; U.S. Pat.No. 4,873,191). The blastocyst is transplanted into the uterus ofanother normal mouse as the foster mother. Then, founder mice (progenymice) are born from the foster mother mouse. By mating the founder micewith normal mice, heterogeneic transgenic mice are obtained. By matingthe heterogeneic transgenic mice with each other, homogenetic transgenicmice are obtained according to Mendel's laws.

Knockout mouse of the present invention is a mouse wherein theendogenous gene encoding the mouse-derived polypeptide of the presentinvention (i.e., “mouse JTT-1 antigen”) has been knocked out(inactivated). It can be prepared, for example, by positive-negativeselection method in which homologous recombination is applied (U.S. Pat.Nos. 5,464,764; 5,487,992; and 5,627,059; Proc. Natl. Acad. Sci. USA86:8932-8935, 1989; Nature 342:435-438, 1989; etc.).

The “antibody” of the present invention can be a polyclonal antibody(antiserum) or a monoclonal antibody, and preferably a monoclonalantibody.

Specifically, it is an antibody reactive to (against, which binds to)the above-mentioned polypeptide or polypeptide fragment of the presentinvention.

The antibody of the present invention can be natural antibodies obtainedby immunizing mammals such as mice, rats, hamsters, guinea pigs, andrabbits with the antigen, such as cells (natural cells, cell lines,tumor cells, etc.) expressing “cell surface molecules” of the presentinvention, transformants overexpressing the polypeptide or cell surfacemolecules of the present invention on the surface thereof prepared usingrecombinant DNA technology on the cell surface, or “polypeptidefragments” or “fusion polypeptides” of the present invention. Theantibody of the present invention also includes chimeric antibodies andhumanized antibodies (CDR-grafted antibodies) that can be produced byrecombinant DNA technology, and human antibodies that can be producedusing human antibody-producing transgenic animals.

The monoclonal antibody includes those having any one isotype of IgG,IgM, IgA, IgD, or IgE. IgG or IgM is preferable.

The polyclonal antibody (antisera) or monoclonal antibody of the presentinvention can be produced by the known methods. Namely, a mammal,preferably, a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig,goat, horse, or cattle, or more preferably, a mouse, rat, hamster,guinea pig, or rabbit is immunized, for example, with an antigenmentioned above with Freund's adjuvant, if necessary.

The polyclonal antibody can be obtained from the antiserum obtained fromthe animal so immunized. In addition, the monoclonal antibodies areproduced as follows. Hybridomas are prepared from the antibody-producingcells obtained from the animal so immunized and myeloma cells that arenot capable of producing autoantibodies. The hybridomas are cloned, andclones producing the monoclonal antibodies showing the specific affinityto the antigen used for immunizing the mammal are screened.

Specifically, the monoclonal antibody can be produced as follows.Immunizations are performed by injecting or implanting once or severaltimes the antigen as mentioned above as an immunogen, if necessary, withFreund's adjuvant, subcutaneously, intramuscularly, intravenously,through the footpad, or intraperitoneally into a non-human mammal,specifically a mouse, rat, hamster, guinea pig, or rabbit, preferably amouse, rat, or hamster (including a transgenic animal generated so as toproduce antibodies derived from another animal such as the transgenicmouse producing human antibody mentioned below). Usually, immunizationsare performed once to four times every one to fourteen days after thefirst immunization. Antibody-producing cells are obtained from themammal so immunized in about one to five days after the lastimmunization. The frequency and interval of immunizations can beappropriately arranged depending on property of the immunogen used.Hybridomas that secrete a monoclonal antibody can be prepared by themethod of Köhler and Milstein (Nature 256:495-497, 1975) and by itsmodified method. Namely, hybridomas are prepared by fusingantibody-producing cells contained in a spleen, lymph node, bone marrow,or tonsil obtained from the non-human mammal immunized as mentionedabove, preferably a spleen, with myelomas without autoantibody-producingability, which are derived from, preferably, a mammal such as a mouse,rat, guinea pig, hamster, rabbit, or human, or more preferably, a mouse,rat, or human.

For example, mouse-derived myeloma P3/X63-AG8.653 (653), P3/NSI/1-Ag4-1(NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2), PAI, F0, orBW5147, rat-derived myeloma 210RCY3-Ag.2.3., or human-derived myelomaU-266AR1, GM1500-6TG-Al-2, UC729-6, CEM-AGR, D1R11, or CEM-T15 can beused as a myeloma used for the cell fusion.

Hybridoma clones producing monoclonal antibodies can be screened bycultivating hybridomas, for example, in microtiter plates and bymeasuring the reactivity of the culture supernatant in the well in whichhybridoma growth is observed, to the immunogen used for the immunizationmentioned above, for example, by enzyme immunoassay such as RIA andELISA.

The monoclonal antibodies can be produced from hybridomas by cultivatingthe hybridomas in vitro or in vivo such as in the ascites fluid of amouse, rat, guinea pig, hamster, or rabbit, preferably a mouse or rat,more preferably mouse and isolating the antibodies from the resultingthe culture supernatant or ascites fluid of a mammal.

Cultivating hybridomas in vitro can be performed depending on theproperty of cells to be cultured, on the object of a test study, and onthe various conditions of a cultivating method, by using known nutrientmedia or any nutrient media derived from known basal media for growing,maintaining, and storing the hybridomas to produce monoclonal antibodiesin culture supernatant.

Examples of basal media are low calcium concentration media such asHam′F12 medium, MCDB153 medium, or low calcium concentration MEM medium,and high calcium concentration media such as MCDB104 medium, MEM medium,D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basalmedia can contain, for example, sera, hormones, cytokines, and/orvarious inorganic or organic substances depending on the objective.

Monoclonal antibodies can be isolated and purified from the culturesupernatant or ascites fluid mentioned above by saturated ammoniumsulfate precipitation, euglobulin precipitation method, caproic acidmethod, caprylic acid method, ion exchange chromatography (DEAE orDE52), affinity chromatography using anti-immunoglobulin column orprotein A column.

Preferable examples of monoclonal antibodies of the present inventionare as follows.

(1) A monoclonal antibody reactive to a polypeptide having an amino acidsequence of SEQ ID NO: 2, a polypeptide fragment derived from thepolypeptide, or a human-derived cell surface molecule composed of thepolypeptide;

(2) A monoclonal antibody reactive to a polypeptide of the presentinvention, a polypeptide fragment derived from the polypeptide, or acell surface molecule composed of the polypeptide, wherein the effect ofthe monoclonal antibody on mitogen-stimulated lymphoblast cells issubstantially the same as the effect of a monoclonal antibody producedby a hybridoma identified by an international deposit accession No. FERMBP-5707 on mitogen-stimulated rat lymphoblast cells; and

(3) A monoclonal antibody reactive to a polypeptide of the presentinvention, a polypeptide fragment derived from the polypeptide, or acell surface molecule composed of the polypeptide, wherein the effect ofthe monoclonal antibody on mitogen-stimulated lymphoblast cells issubstantially the same as the effect of a monoclonal antibody producedby a hybridoma identified by an international deposit accession No. FERMBP-5708 on mitogen-stimulated rat lymphoblast cells.

In addition, the monoclonal antibody of the present invention includesthat produced by the hybridoma identified by an international depositaccession No. FERM BP-5707 or No. FERM BP-5708.

The “chimeric monoclonal antibody” of the present invention is amonoclonal antibody prepared by genetic engineering, and specificallymeans a chimeric antibody such as mouse/human chimeric monoclonalantibody whose variable regions are derived from immunoglobulin of annon-human mammal (mouse, rat, hamster, etc.) and whose constant regionsare derived from human immunoglobulin.

The constant region derived from human immunoglobulin has the amino acidsequence inherent in each isotype such as IgG (IgG1, IgG2, IgG3, IgG4),IgM, IgA, IgD, and IgE. The constant region of the recombinant chimericmonoclonal antibody of the present invention can be that of humanimmunoglobulin belonging to any isotype. Preferably, it is the constantregion of human IgG.

The chimeric monoclonal antibody of the present invention can beproduced, for example, as follows. Needless to say, the productionmethod is not limited thereto.

A mouse/human chimeric monoclonal antibody can be prepared, referring toExperimental Medicine: SUPPLEMENT, Vol. 1.6, No.10 (1988); and examinedpublished Japanese patent application (JP-B) No. Hei 3-73280. Namely, itcan be prepared by operably inserting CH gene (C gene encoding theconstant region of H chain) obtained from the DNA encoding humanimmunoglobulin downstream of active VH genes (rearranged VDJ geneencoding the variable region of H chain) obtained from the DNA encodinga mouse monoclonal antibody isolated from the hybridoma producing themouse monoclonal antibody, and CL gene (C gene encoding the constantregion of L chain) obtained from the DNA encoding human immunoglobulindownstream of active VL genes (rearranged VJ gene encoding the variableregion of L chain) obtained from the DNA encoding the mouse monoclonalantibody isolated from the hybridoma, into the same or different vectorsso as for them to be expressed, following by transforming host cellswith the expression vector, and then by cultivating the transformants.

Specifically, DNAs are first extracted from mouse monoclonalantibody-producing hybridomas by the usual method, digested withappropriate restriction enzymes (for example, EcoRI and HindIII),electrophoresed (using, for example, 0.7% agarose gel), and analyzed bySouthern blotting. After an electrophoresed gel is stained, for example,with ethidium bromide and photographed, the gel is given with markerpositions, washed twice with water, and soaked in 0.25 M HCl for 15minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minuteswith gently stirring. The DNAs are transferred to a filter for 4 hoursby the usual method. The filter is recovered and washed twice with2×SSC. After the filter is sufficiently dried, it is baked at 75° C. for3 hours. After baking, the filter is treated with 0.1×SSC/0.1% SDS at65° C. for 30 minutes. Then, it is soaked in 3×SSC/0.1% SDS. The filterobtained is treated with prehybridization solution in a plastic bag at65° C. for 3 to 4 hours.

Next, ³²P-labeled probe DNA and hybridization solution are added to thebag and reacted at 65° C. about 12 hours. After hybridization, thefilter is washed under appropriate salt concentration, reactiontemperature, and time (for example, 2×SSC-0.1% SDS, room temperature, 10minutes). The filter is put into a plastic bag with a little 2×SSC, andsubjected to autoradiography after the bag is sealed.

Rearranged VDJ gene and VJ gene encoding H chain and L chain of a mousemonoclonal antibody are identified by Southern blotting mentioned above.The region comprising the identified DNA fragment is fractioned bysucrose density gradient centrifugation and inserted into a phage vector(for example, Charon 4A, Charon 28, XEMBL3, XEMBL4, etc.). E. coli (forexample, LE392, NM539, etc.) is transformed with the phage vector togenerate a genomic library. The genomic library is screened by plaquehybridization such as Benton-Davis method (Science 196:180-182, 1977)using appropriate probes (H chain J gene, L chain (K) J gene, etc.) toobtain positive clones comprising rearranged VDJ gene or VJ gene. Bymaking the restriction map and determining the nucleotide sequence ofthe clones obtained, it is confirmed that genes comprising the desired,rearranged VH (VDJ) gene or VL (VJ) gene are obtained.

Separately, human CH gene and human CL gene used for chimerization areisolated. For example, when a chimeric antibody with human IgG1 isproduced, CΥ1 gene as a CH gene, and Cκ gene as a CL gene, are isolated.These genes can be isolated from human genomic library with mouse CΥ1gene and mouse Cκ gene, corresponding to human CΥ1 gene and human Cκgene, respectively, as probes, taking advantage of high homology betweenthe nucleotide sequences of mouse immunoglobulin gene and that of humanimmunoglobulin gene.

Specifically, DNA fragments comprising human Cκ gene and an enhancerregion are isolated from human λ Charon 4A HaeIII-AluI genomic library(Cell 15:1157-1174, 1978), for example, with a 3 kb HindIII-BamHIfragment of clone Ig146 (Proc. Natl. Acad. Sci. USA 75:4709-4713, 1978)and a 6.8 kb EcoRI fragment of clone MEP10 (Proc. Natl. Acad. Sci. USA78:474-478, 1981) as probes. In addition, for example, after human fetalhepatocyte DNA is digested with HindIII and fractioned by agarose gelelectrophoresis, a 5.9 kb fragment is inserted into λ788 and then humanCγ1 gene is isolated with the probes mentioned above.

Using mouse VH gene, mouse VL gene, human CH gene, and human CL gene soobtained, and taking promoter region and enhancer region intoconsideration, human CH gene is inserted downstream mouse VH gene andhuman CL gene is inserted downstream mouse VL gene into an expressionvector such as pSV2gpt or pSV2neo with appropriate restriction enzymesand DNA ligase by the usual method. In this case, chimeric genes ofmouse VH gene/human CH gene and mouse VL gene/human CL gene can berespectively inserted in the same expression vector or in differentexpression vectors.

Chimeric gene-inserted expression vector(s) thus prepared are introducedinto myelomas that do not produce antibodies, for example, P3X63∘Ag8∘653cells or SP210 cells by protoplast fusion method, DEAE-dextran method,calcium phosphate method, or electroporation method. The transformantsare screened by cultivating in media containing a drug corresponding tothe drug resistance gene inserted into the expression vector and, then,cells producing desired chimeric monoclonal antibodies are obtained.

Desired chimeric monoclonal antibodies are obtained from the culturesupernatant of antibody-producing cells thus screened.

The “humanized monoclonal antibody (CDR-grafted antibody)” of thepresent invention is a monoclonal antibody prepared by geneticengineering and specifically means a humanized monoclonal antibodywherein a portion or the whole of the complementarity determiningregions of the hypervariable region are derived from the complementaritydetermining regions of the hypervariable region from a monoclonalantibody of an non-human mammal (mouse, rat, hamster, etc.), theframework regions of the variable region are derived from the frameworkregions of the variable region from human immunoglobulin, and theconstant region is derived from human a constant region fromimmunoglobulin.

The complementarity determining regions of the hypervariable regionexists in the hypervariable region in the variable region of an antibodyand means three regions which directly and complementary binds to anantigen (complementarity-determining residues, CDR1, CDR2, and CDR3).The framework regions of the variable region means four comparativelyconserved regions lying upstream, downstream or between the threecomplementarity determining regions (framework region, FR1, FR2, FR3,and FR4).

In other words, a humanized monoclonal antibody means that in which thewhole region except a portion or the whole of the complementaritydetermining regions of the hypervariable region of a nonhumanmammal-derived monoclonal antibody have been replaced with theircorresponding regions derived from human immunoglobulin.

The constant region derived from human immunoglobulin has the amino acidsequence inherent in each isotype such as IgG (IgG1, IgG2, IgG3, IgG4),IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonalantibody in the present invention can be that from human immunoglobulinbelonging to any isotype. Preferably, it is the constant region of humanIgG. The framework regions of the constant region derived from humanimmunoglobulin are not particularly limited.

The humanized monoclonal antibody of the present invention can beproduced, for example, as follows. Needless to say, the productionmethod is not limited thereto.

For example, a recombinant humanized monoclonal antibody derived frommouse monoclonal antibody can be prepared by genetic engineering,referring to unexamined Japanese patent publication (JP-WA) No. Hei4-506458 and unexamined Japanese patent publication (JP-A) No. Sho62-296890. Namely, at least one mouse H chain CDR gene and at least onemouse L chain CDR gene corresponding to the mouse H chain CDR gene areisolated from hybridomas producing mouse monoclonal antibody, and humanH chain gene encoding the whole regions except human H chain CDRcorresponding to mouse H chain CDR mentioned above and human L chaingene encoding the whole region except human L chain CDR correspond tomouse L chain CDR mentioned above are isolated from human immunoglobulingenes.

The mouse H chain CDR gene(s) and the human H chain gene(s) so isolatedare operably inserted into an appropriate vector so that they can beexpressed. Similarly, the mouse L chain CDR gene(s) and the human Lchain gene(s) are operably inserted into another appropriate vector sothat they can be expressed. Alternatively, the mouse H chain CDRgene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human Lchain gene(s) can be operably inserted into the same expression vectorso that they can be expressed. Host cells are transformed with theexpression vector thus prepared to obtain transformants producinghumanized monoclonal antibody. By cultivating the transformants, desiredhumanized monoclonal antibody is obtained from culture supernatant.

The “human monoclonal antibody” of the present invention isimmunoglobulin in which the entire regions comprising the variable andconstant region of H chain, and the variable and constant region of Lchain constituting immunoglobulin are derived from the gene encodinghuman immunoglobulin.

The human antibody can be produced in the same way as the productionmethod of polyclonal or monoclonal antibodies mentioned above byimmunizing, with an antigen, a transgenic animal which for example, atleast human immunoglobulin gene(s) have been integrated into the locusof a non-human mammal such as a mouse by the usual method.

For example, a transgenic mouse producing human antibodies is preparedby the methods described in Nature Genetics 7:13-21, 1994; NatureGenetics 15:146-156, 1997; JP-WA Nos. Hei 4-504365 and Hei 7-509137;Nikkei Science 6:40-50, 1995; International patent publication No.W094/25585; Nature 368:856-859, 1994; and JP-WA No. Hei 6-500233.

In addition, recently developed technique for producing a human-derivedprotein from the milk of a transgenic cow or pig can also be applied(Nikkei Science, pp. 78-84, Apr., 1997).

The “portion of an antibody” used in the present invention means apartial region of the monoclonal antibody as mentioned above, andspecifically, means F(ab′)₂, Fab′, Fab, Fv (variable fragment ofantibody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domainantibody) (Exp. Opin. Ther. Patents 6:441-456, 1996).

“F(ab′)₂” and “Fab′” can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andmeans an antibody fragment generated by digesting immunoglobulin nearthe disulfide bonds existing between the hinge regions in each of thetwo H chains. For example, papain cleaves IgG upstream of the disulfidebonds existing between the hinge regions in each of the two H chains togenerate two homologous antibody fragments in which an L chain composedof VL (L chain variable region) and CL (L chain constant region), and anH chain fragment composed of VH (H chain variable region) and CHγ1 (γ1region in the constant region of H chain) are connected at their Cterminal regions through a disulfide bond. Each of such two homologousantibody fragments is called Fab′. Pepsin also cleaves IgG downstream ofthe disulfide bonds existing between the hinge regions in each of thetwo H chains to generate an antibody fragment slightly larger than thefragment in which the two above-mentioned Fab′ are connected at thehinge region. This antibody fragment is called F(ab′)₂.

The “pharmaceutical composition” of the present invention comprises anyone of the “polypeptides” of the present invention as defined above;“homodimer molecule”, “polypeptide fragment”, “fusion polypeptide”comprising the polypeptide; “homodimer molecule” comprising the fusionpolypeptides, “antibody”, or “portion of an antibody”; and apharmaceutically acceptable carrier.

The “pharmaceutically acceptable carrier” includes a excipient, adiluent, an expander, a decomposition agent, a stabilizer, apreservative, a buffer, an emulsifier, an aromatic, a colorant, asweetener, a viscosity increasing agent, a flavor, a solubilityincreasing agent, or other additives. Using one or more of suchcarriers, a pharmaceutical composition can be formulated into tablets,pills, powders, granules, injections, solutions, capsules, troches,elixirs, suspensions, emulsions, or syrups. The pharmaceuticalcomposition can be administered orally or parenterally. Other forms forparenteral administration include a solution for external application,suppository for rectal administration, and pessary, prescribed by theusual method, which comprises one or more active ingredient.

The dosage can vary depending on the age, sex, weight, and symptom of apatient, effect of treatment, administration route, period of treatment,or the kind of active ingredient (polypeptide or antibody mentionedabove) contained in the pharmaceutical composition. Usually, thepharmaceutical composition can be administered to an adult in a dose of10 μg to 1000 mg (or 10 μg to 500 mg) per one administration. Dependingon various conditions, the dosage less than that mentioned above may besufficient in some cases, and the dosage more than that mentioned abovemay be necessary in other cases.

In particular, the injection can be produced by dissolving or suspendingthe antibody in a non-toxic, pharmaceutically acceptable carrier such asphysiological saline or commercially available distilled water forinjection with adjusting a concentration to 0.1 μg antibody/ml carrierto 10 mg antibody/ml carrier. The injection thus produced can beadministered to a human patient in need of treatment in a dose of 1 μgto 100 mg/kg body weight, preferably 50 μg to 50 mg/kg body weight onceor more times a day. Examples of administration route are medicallyappropriate administration routes such as intravenous injection,subcutaneous injection, intradermal injection, intramuscular injection,or intraperitoneal injection, preferably intravenous injection.

The injection can also be prepared into a non-aqueous diluent (forexample, propylene glycol, polyethylene glycol, vegetable oil such asolive oil, and alcohol such as ethanol), suspension, or emulsion.

The injection can be sterilized by filtration with abacteria-non-penetrated filter, by mixing bactericide, or byirradiation. The injection can be produced in the form that is preparedupon use. Namely, it is freeze-dried to be a sterile solid composition,and can be dissolved in sterile distilled water for injection or anothersolvent before use.

The pharmaceutical composition of the present invention can be appliedto treating or preventing various autoimmune diseases, allergicdiseases, or inflammatory diseases caused by the activation oflymphocytes such as T cells and the regulation of activated lymphocytefunctions. Examples of the diseases are rheumatoid arthritis, multiplesclerosis, autoimmune thyroiditis, allergic contact dermatitis, chronicinflammatory dermatosis such as lichen planus, systemic lupuserythematosus, insulin dependent diabetes mellitus, and psoriasis.

The therapeutic effect of the pharmaceutical composition of the presentinvention for symptom of various diseases can be tested by the usualmethod by administering it to an known disease model animal.

Examples of the model include (1) a (NZB/NZW)F1 mouse, a model for humansystemic lupus erythematosus (SLE) (Science 125:1225-1227, 1994); (2)experimental allergic encephalomyelitis (EAE), a model for multiplesclerosis (MS) (J. Clin. Invest. 95:2783-2789, 1995); (3) an NOD(non-obese diabetes) mouse, a model for insulin dependent diabetesmellitus (IDDM) (J. Exp. Med. 181:1145-1155, 1995); (4) rat nephritismodel by renal glomerulus basement membrane immunity, Goodpasture'snephritis model (Eur. J. Immunol. 24:1249-1254, 1994); and (5) a DBA/1mouse, a model for human rheumatoid arthritis (Eur. J. Immunol.26:2320-2328, 1996).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are micrographs showing the state of aggregation of FTL435 cellsinduced by “JTT-1 antibody” and the state of inhibition of the cellaggregation by “JTT.2 antibody.”

Subfigure (a) shows the state of the cells in the absence of anyhybridoma supernatant, subfigure (b) shows the state of cell aggregationinduced by “JTT-1 antibody,” subfigure (c) shows the state of the cellaggregation in the presence of “anti-ICAM-1 antibody” together with“JTT-1 antibody,” and subfigure (d) shows the state of the cellaggregation in the presence of “JTT-2 antibody” together with “JTT-1antibody.”

FIG. 2 are micrographs showing the state of aggregation of FTL435 cellsand rat activated lymphoblasts induced by “JTT-1 antibody” and the stateof inhibition of the cell aggregation by “JTT.2 antibody.”

Subfigure (a) shows the state of FTL435 cells in the absence of anyantibody, subfigure (b) shows the state of FTL435 cells in the presenceof PMA, subfigure (c) shows the state of FTL435 cells in the presence of“JTT-1 antibody,” subfigure (d) shows the state of FTL435 cells in thepresence of anti-LFA-l antibody together with “JTT-1 antibody,”subfigure (e) shows the state of FTL435 cells in the presence ofanti-CD18 antibody together with “JTT-1 antibody,” subfigure (f) showsthe state of FTL435 cells in the presence of anti-ICAM-1 antibodytogether with “JTT-1 antibody,” subfigure (g) shows the state ofactivated lymphoblasts in the absence of any antibody, subfigure (h)shows the state of activated lymphoblasts in the presence of PMA,subfigure (i) shows the state of activated lymphoblasts in the presenceof “JTT-1 antibody,” subfigure (j) shows the state of activatedlymphoblasts in the presence of anti-LFA-1 antibody together with “JTT-1antibody,” subfigure (k) shows the state of activated lymphoblasts inthe presence of anti-CD18 antibody together with “JTT-1 antibody,” andsubfigure (1) shows the state of activated lymphoblasts in the presenceof anti-ICAM-1 antibody together with “JTT-1 antibody.”

FIG. 3 shows the expression state of “JTT-1 antigen” and “JTT-2 antigen”in various cells measured with a flow-cytometer.

FIG. 4 shows the expression state of “JTT-1 antigen” in variouslymphocytic cells measured with a flow-cytometer.

FIG. 5 is a photograph showing electrophoretogram of “JTT-1 antigen”analyzed by SDS-PAGE.

FIG. 6 are micrographs showing the state of adhesion of rat thymocytesto the microtiter plate coated with purified “JTT-1 antigen,” where theadhesion is induced in the presence of “JTT-1 antibody,” and the stateof inhibition of the cell adhesion by “JTT-2 antibody.”

Subfigure (a) shows the state of adhesion of the cells to the platewhich has not been coated with “JTT-1 antigen,” subfigure (b) shows thestate of adhesion of the cells to the plate coated with “JTT-1 antigen”in the absence of any antibody, subfigure (c) shows the state ofadhesion of the cells to the plate coated with “JTT-1 antigen” in thepresence of the Fab fragments of “JTT-1 antibody,” and subfigure (d)shows the state of adhesion of the cells to the plate coated with “JTT-1antigen” in the presence of “JTT-2 antibody” together with the Fabfragments of “JTT-1 antibody.”

FIG. 7 shows the relative cell number of thymocytes adhering to theplate coated with purified “JTT-1 antigen” measured in terms offluorescence intensity. “Ag(−)” shows the relative cell number in theplate which has not been coated with “JTT-1 antigen,” “Ag(+)” shows therelative cell number in the plate coated with “JTT-1 antigen” in theabsence of any antibody, “Ag(+)+JTT-1 Fab” shows the relative cellnumber in the plate coated with “JTT-1 antigen” in the presence of theFab fragments of “JTT-1 antibody”, and “Ag(+)+JTT-1 Fab+JTT-2” shows therelative cell number in the plate coated with “JTT-1 antigen” in thepresence of “JTT-2 antibody” together with the Fab fragments of “JTT-1antibody.”

FIG. 8 shows the expression state of “rat JTT-1 antigen” and “rat JTT-2antigen” in COS cells transformed with cDNA encoding “rat JTT-1 antigen”with a flow-cytometer.

FIG. 9 shows the structural characteristics of amino acid sequence of“JTT-1 antigen” revealed by hydropathy plot analysis.

FIG. 10 shows the homology among amino acid sequences of human (SEQ IDNO:2), rat (SEQ ID NO:13), and mouse “JTT-1 antigen” (SEQ ID NO:14) and“rat JTT-1 antigen” mutant (SEQ ID NO:15) (the consensus sequence islisted as SEQ ID NO:16).

FIG. 11 shows the homology among amino acid sequences and conservationstate of motifs in “human JTT-1 antigen,” “human CD28 molecule”, and“human CTLA-4 molecule.” (SEQ ID NOs:2, 25, and 26, respectively) (theconsensus sequence is listed as SEQ ID NO:17).

FIG. 12 schematically shows the protein secondary structure of, andtheir similarity among “human JTT-1 antigen;” (SEQ ID NOs:21 and 22),“human CD28 molecule,” (SEQ ID NOs:18 and 19), and “human CTLA-4molecule.” (SEQ ID NOs:18 and 20).

FIG. 13 schematically shows the structure of the genomic DNA encoding“mouse JTT-1 antigen.”

FIG. 14 shows the difference in amino acid sequences between “rat JTT-1antigen” and its alternative splicing mutant (SEQ ID NOs:13 and 15,respectively) (the consensus sequence is listed as SEQ ID NO:23).

FIG. 15 shows the degree of the growth of human peripheral bloodlymphocytes induced by the monoclonal antibody against “human JTT-1antigen,” where the degree of the growth was measured by [³H] thymidineuptake.

The ordinate shows the amount of uptake (dpm) of [³H] thymidine into thecells.

FIG. 16 shows the therapeutic effect of the monoclonal antibody against“JTT-1 antigen” on experimental allergic encephalomyelitis (EAE) in adisease model rat.

The ordinate shows the scored degree of disease symptom, and theabscissa shows the days after immunization for induction of EAE.

FIG. 17 shows the therapeutic effect of the monoclonal antibody against“JTT-1 antigen” on glomerulonephritis in a disease model rat.

The ordinate shows the amount of urinary excretion of proteins, and theabscissa shows the time course (week) after immunization for inductionof glomerulonephritis

FIG. 18 shows a column histogram in purification of fusion polypeptidebetween “rat JTT-1 antigen” extracellular region and human IgFc(rJTT-1-IgFc) with protein A Sepharose column.

FIG. 19 is a photograph showing electrophoretogram of rJTT-1-IgFcanalyzed by SDS-PAGE.

FIG. 20 shows a column histogram in purification of fusion polypeptidebetween “human JTT-1 antigen” extracellular region and human IgFc(hJTT-1-IgFc) with protein A Sepharose column.

FIG. 21 is a photograph showing electrophoretogram of hJTT-1-IgFcanalyzed by SDS-PAGE.

FIG. 22 schematically shows the structure of the gene transfer(targeting) vector used for preparation of a transgenic mouse into whichthe cDNA encoding “rat JTT-1 antigen” has been introduced.

BEST MODE FOR IMPLEMENTING THE INVENTION

The present inventions are described in more detail with reference toExamples below, but are not to be construed to be limited thereto.

Example 1 Preparation of Monoclonal Antibodies

Antibody-producing hybridomas were prepared according to the method ofKöhler et al. (Omori et al., Blood, 81:101-111, 1993), and monoclonalantibodies were prepared according to the method of Kannagi et al.(Handbook of Experimental Immunology, 4:117.21-117.21, 1986).

First, rat thymoma cell line FTL435 cells were administered as animmunizing antigen to BALB/c mice into their footpad in an amount of 10⁷cells/mouse at intervals of 0, 7, 14, and 28 days. The mixture of theantigen with Freund's complete adjuvant was administered only in thefirst immunization. Two days after the last immunization, the lymphnodes of the mice were taken out and fused with mouse myeloma cells PAI(JCR No. B0113; Stocker et al., Res. Disclosure, 217:155, 1982) by theusual method to obtain many hybridomas producing monoclonal antibodies.

Example 2 Screening of Hybridomas and Characterization of MonoclonalAntibodies

The hybridomas prepared in Example 1 were screened by analyzing theeffect of the antibodies produced in the culture supernatant of thehybridomas on FTL435 cells, which were used as the immunogen. FTL435cells (5×10⁶ cells/ml, 0.1 ml) were seeded into each well of a 96-wellmicrotiter plate and cultivated at 37° C. for an hour in the presence ofculture supernatant of each hybridoma (10 μg/ml each). The resultsobtained for hybridoma clones “JTT-1” and “JTT-2” are shown in FIG. 1and FIG. 2.

It was revealed that a monoclonal antibody produced by hybridoma clone“JTT-1” (“JTT-1 antibody”) strongly agglutinated FTL435 cells (FIG. 1(b) and FIG. 2( c)) and that addition of “JTT-2 antibody” stronglyinhibited the aggregation of FTL435 cells induced by “JTT-1 antibody”stimulation (FIG. 1( d)). The assays, in which no hybridoma supernatantwas added, were used as controls (FIG. 1( a) and FIG. 2( a)).

In order to determine whether the aggregation of FTL435 cells induced by“JTT-1 antibody” stimulation was caused by the cell adhesion betweenintercellular adhesion molecule-1 (ICAM-1) and lymphocytefunction-associated antigen-1 (LFA-1), which is a representative knownpathway of cell adhesion, FTL435 cells were cultivated at 37° C. for anhour in the presence of anti-rat ICAM-1 antibody 1A29 (10 μg/ml; IgG1)or anti-rat LFA-1 antibody (10 μg/ml; IgG2a) together with “JTT-1antibody.”

The aggregation of FTL435 cells by “JTT-1 antibody” stimulation wasinhibited by neither anti-ICAM-1 antibody nor anti-LFA-1 antibody(anti-ICAM-1 antibody, FIG. 1( c) and FIG. 2( f); anti-LFA-1 antibody,FIG. 2( d)).

In order to further analyze the cell agglutination ability of “JTT-1antibody,” the ability to agglutinate rat lymphoblast cells activatedwith concanavalin A stimulation was analyzed in the same manner asmentioned above. The results are shown in FIG. 2.

Similar to the effect on FTL435 cells, the aggregation of activatedlymphoblast cells was induced by “JTT-1 antibody” stimulation (FIG. 2(i)). The aggregation of activated lymphoblast cells by “JTT-1 antibody”stimulation was mostly inhibited by anti-LFA-1 antibody (FIG. 2( j)) andanti-ICAM-1 antibody (FIG. 2( l)). (However, partial aggregationoccurred.)

As understood from the control assay (FIG. 2( g)), in which no antibodywas added, activated lymphocytes such as activated lymphoblasts showedno aggregation through cell adhesion unless they receive the stimulationwith phorbol myristate acetate (PMA, which activates LFA-1) (FIG. 2( h))or “JTT-1 antibody” (FIG. 2( i)). Therefore, the fact that anti-LFA-1antibody partially inhibited cell aggregation by “JTT-1 antibody”stimulation indicates that LFA-1 in activated lymphoblast cells wasactivated by “JTT-1 antibody” stimulation. This also indicates thatmolecules recognized by “JTT-1 antibody” are involved in some signaltransmission.

Hybridoma clones “JTT-1” and “JTT-2” have been deposited under theBudapest Treaty with international depository authority, NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry,Japan (1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) since Oct. 11, 1996with an international accession Nos. FERM BP-5707 and FERM BP-5708,respectively.

Analysis using mouse monoclonal antibody isotype identification kit(Amersham) determined that the isotype of monoclonal antibodies producedfrom each hybridoma (JTT-1 antibody and JTT-2 antibody) were both IgGl.

Example 3 Reactivity of “JTT-1 Antibody” and “JTT-2 Antibody” to VariousCells

In order to analyze the expression pattern of molecules recognized by“JTT-1 antibody” and “JTT-2 antibody” in various cells, the reactivitiesof the antibodies to various cells were examined. Molecules recognizedby “JTT-1 antibody” or “JTT-2 antibody” are designated “JTT-1 antigen”or “JTT-2 antigen”, respectively.

A five- to ten-week-old Wistar rat (150 to 250 g) was killed byanesthesia with diethyl ether. The thymus and spleen were taken out ofits chest and abdomen, respectively, by celiotomy, and homogenized toprepare cell suspension. The resulting spleen cells were cultivated inRPMI1640 medium containing 2 μg/ml concanavalin A and 10% FCS at 37° C.for 3 days to prepare activated lymphoblasts.

FTL435 cells, thymocytes, spleen cells, and activated lymphoblasts(5×10⁵ cells each) were reacted with “JTT-1 antibody” or “JTT-2antibody” and then with FITC-labeled anti-mouse IgG (Cappel). Thefluorescence intensity of the stained cells was measured withEPICS-Elite flow cytometer.

The results are shown in FIG. 3. In FTL435 cells, the strong expressionof each “JTT-1 antigen” and “JTT-2 antigen” was observed. While theantigens were expressed in thymocytes, they were expressed only a littlein spleen cells. However, in activated lymphoblasts obtained bysimulating spleen cells with concanavalin A, “JTT-1 antigen” and “JTT-2antigen” were strongly expressed. In addition, in each kind of cells,the expression pattern of “JTT-1 antigen” and “JTT-2 antigen” coincidedwith each other. These results indicate that “JTT-1 antigen” and “JTT-2antigen” are the same molecules.

Example 4 Reactivity of “JTT-1 Antibody” to Various Lymphocytic Cells

In order to analyze the expression pattern of molecules (“JTT-1antigen”) recognized by “JTT-1 antibody” in various lymphocytic cells,the reactivity of “JTT-1 antibody” to lymph nodes, T lymphoblastsderived from spleen, and B lymphoblasts derived from spleen of two kindsof rats (Wistar rat and F344 rat) was analyzed.

A five- to ten-week-old Wistar rat and F344 rat (150 to 250 g) werekilled by anesthesia with diethyl ether. The lymph nodes and spleen weretaken out of each rat by celiotomy, and homogenized to prepare cellsuspension. The resulting cell suspension from spleen was cultivated inRPMI1640 medium containing 2 μg/ml concanavalin A (ConA) and 10% FCS at37° C. for 3 days. Activated T lymphoblasts and activated B lymphoblastswere obtained from each rat after 1-day and 3-day cultivation. Inaddition, spleen-derived T lymphoblasts and B lymphoblasts obtainedbefore lymph node cells and ConA were added were used as controls.

Each cells (5×10⁵ cells each) were reacted with biotin-labeled anti-ratT cell antibody or biotin-labeled anti-rat B cell antibody (10 μg/ml,Seikagaku Corporation), and subsequently with phycoerythrin-labeledstreptavidin. The cells were then reacted with 10 μg/ml FITC-labeled“JTT-1 antibody.” The fluorescence intensity of the stained cells wasmeasured with EPICS-Elite flow cytometer.

The results are shown in FIG. 4. In both activated T lymphoblasts andactivated B lymphoblasts from Wistar rat and F344 rat, the strongexpression of “JTT-1 antigen” was observed from day 1 of activation withConA stimulation. In addition, the expression pattern of “JTT-1 antigen”in each kind of cells almost perfectly coincided with each other.

Example 5 Characterization of “JTT-1 Antigen” and “JTT-2 Antigen” byImmunoprecipitation

“JTT1 antigen” and “JTT-2 antigen” were characterized byimmunoprecipitation using FTL435 cells.

(1) Preparation of Biotinylated Soluble Cell Surface Molecules

FTL435 cells were washed with PBS, suspended in physiological salinecontaining 100 μg/ml NHS-biotin and 0.1 M HEPES (pH 8.0) to adjust 1×10⁷cells/ml, and incubated at room temperature for 40 minutes. The cellswere washed three times with PBS, lysis buffer (1% NP-40, 10 mM Tris-HCl(pH 7.4), 0.15 M NaCl) was added thereto to adjust 5×10⁷ cells/ml, andthe mixture was allowed to react at 4° C. for 30 minutes to lyse thecells. The cell lysate obtained was centrifuged, and the supernatantcomprising biotinylated soluble cell surface molecules was stored at−80° C.

(2) Immunoprecipitation and SDS-PAGE Analysis

The purified sample of “JTT-1 antibody” purified by the usual methodfrom the culture supernatant of the hybridoma clone “JTT-1” prepared inExample 1 was mixed with protein G-Sepharose beads to adjust 2 mg/ml,and allowed to react at 4° C. for an hour to bind the antibody with thebeads. After the beads were washed, 500 μl of the biotinylated FTL435cell lysate was added to 10 μl of the beads, and the mixture was allowedto react at 4° C. for 2 hours. After the beads were washed with lysisbuffer three times, 50 μl of glycanase buffer (sodium phosphate buffer(pH 7.0) containing 0.15% SDS) was added to the beads, and the mixturewas boiled to elute the bound molecules trapped by the antibody-boundbeads. 1.25% NP-40 and 20 U/ml N-glycanase were added to a fraction ofthe sample so eluted, and the mixture was allowed to react overnight todigest N-linked sugar chains.

An equal volume of sample buffer (Enprotech) for SDS-PAGE was added to 5μl of the eluted sample in the presence or absence of 2-mercaptoethanol,and the mixture was boiled. After electrophoresis, the gel wastransferred to a PVDF membrane. The membrane was blocked with 3% BSA-PBSand reacted with peroxidase-labeled streptavidin to detect biotinylatedsoluble cell surface molecules trapped by “JTT-1 antibody” with ECLsystem (Amersham) as described in the manual.

The results are shown in FIG. 5. The “JTT-1 antibody”-recognizedmolecule (“JTT-1 antigen”) on FTL435 cells showed the molecular weightof about 47 kD under the non-reduced conditions (“(−)” in FIG. 5) andabout 24 kD and 28 kD under the reduced conditions (“(+)” in FIG. 5). Asthe result of digestion of N-linked sugar chains (“+N-gly” in FIG. 5),“JTT-1 antigen” was converged on a single band of about 36 kD under thenon-reduced conditions and about 20 kD under the reduced conditions.These results suggest that “JTT-1 antigen” forms a dimer in which thesame core proteins have different sugar chains. Completely the sameresults were obtained in the experiment performed as mentioned aboveusing “JTT-2 antibody.” Considering these results together with theresults of Example 3 and Example 7 below, “JTT-1 antigen” (moleculerecognized by “JTT-1 antibody”) and “JTT-2 antigen” (molecule recognizedby “JTT-2 antibody”) have been thought to be identical to each other.

Example 6 Adhesion Experiment of Rat Thymocytes to Purified “JTT-1Antigen” and N Terminal Amino Acid Analysis

The following experiments were performed to analyze whether the moleculethat “JTT-1 antibody” recognizes (“JTT-1 antigen”) functions as anadhesion molecule. N-terminal amino acid analysis was also performed.

(1) Preparation of “JTT-1 Antibody”-Affinity Column

The purified sample (2 mg in 2 ml) of “JTT-1 antibody” purified by theusual method from the culture supernatant of the hybridoma clone “JTT-1”prepared in Example 1 was mixed with 1 ml of protein G- Sepharose resin,and the mixture was allowed to react at 4° C. for an hour. The resin waswashed three times with 200 mM triethanolamine (pH 8.2). The resin wasthen incubated in triethanolamine (pH 8.2) containing 10 mM dimethylpimelimidate (DMP) at room temperature for an hour to covalently bind“JTT-1 antibody” to the resin.

(2) Purification of “JTT-1 Antigen”

FTL435 cells were cultivated in RPMI1640 medium containing 10% FCS. Thecells were harvested by centrifugation to obtain a pellet and washedwith PBS three times. Lysis buffer (1% NP-40, 10 mM Tris-HCl (pH 7.4),0.15 M NaCl) was added to the washed pellet to adjust 5×10⁷ cells/ml,and the mixture was allowed to react at 4° C. for 30 minutes to lyse thecells. The cell lysate obtained was centrifuged, and the supernatantcontaining soluble cell surface molecules was stored at −80° C.

The lysate (400 ml) was loaded onto “JTT-1 antibody”-affinity column.After the column was washed with 50 ml of the lysis buffer and 20 ml ofPBS, “JTT-1 antigen” was eluted with 0.2 M glycine buffer (pH 2.8). 1 MTris buffer was added to the “JTT-1 antigen” so eluted forneutralization. “JTT-1 antigen” obtained was stored at −80° C.

(3) Determination of N Terminal Amino Acid Sequence

After the purified “JTT-1 antigen” was subjected to SDS-PAGE, theN-terminal amino acid sequence was determined by the usual method. Theresult revealed that “JTT-1 antigen”contained an amino acid sequenceGlu-Leu-Asn-Asp-Leu-Ala-Asn-His-Arg (amino acid residues 21-29 of SEQ IDNO:13).

(4) Adhesion Experiment

A five- to ten-week-old Wistar rat (150 to 250 g) was killed byanesthesia with diethyl ether. The thymus was taken out of its chest byceliotomy and homogenized to prepare thymocyte suspension. 10μ′,7′-bis(carboxyethyl)carboxyfluorescein tetraacetoxy-methyl ester(BCECF-AM; Molecular Probes) was added to the suspension, and themixture was incubated at 37° C. for 30 minutes to fluorescently labelthe thymocytes. The cells were washed with PBS and suspended in RPMI1640medium containing 10% FCS to adjust 2×10⁷ cells/ml.

The purified “JTT-1 antigen” obtained in (2) was coated on a 96-wellELISA plate at the concentration of 10 μl/well overnight. After theplate was washed with PBS, 200 μl/well of PBS containing 3% BSA wasadded to the plate, and blocking was performed for 2 hours. After theplate was washed with PBS, (1) only fluorescence-labeled thymocytes(2×10⁷ cells/ml, 0.1 ml); (2) fluorescence-labeled thymocytes (sameconcentration) and “JTT-1 antibody” Fab fragments prepared by the usualmethod (5μ “JTT-1 antibody” Fab fragments (same concentration), and“JTT-2 antibody” (10μ ° C. for an hour. In order to remove unboundcells, each well was washed once with RPMI1640 medium containing 10%FCS. Each well was observed with light microscope. Then, 100 μl of 0.1%NP-40 was added to each well, and the cells adhered to the plate werelysed. The relative cell number of fluorescence-labeled thymocytesadhered to each well was counted by measuring the fluorescence intensityat 538 nm (excited at 485 nm) with Fluoroscan II Microplate Fluorometer(Flow Laboratories). The assay in which a plate was not coated withpurified “JTT-1 antigen” was used as a control.

The results of light microscopy observation are shown in FIG. 6.

Thymocytes significantly adhered to purified “JTT-1 antigen” only in thepresence of “JTT-1 antibody” Fab fragments (FIG. 6( c)). The adhesionwas significantly inhibited by “JTT-2 antibody” (FIG. 6( d)).

FIG. 7 shows the relative cell number of thymocytes adhered to “JTT-1antigen” coated on each well in terms of fluorescent intensity.

From these results, it was revealed that “JTT-1 antigen” functions as anadhesion molecule.

Example 7 Cloning of cDNA Encoding Rat “JTT-1 Antigen”

1. Preparation of cDNA Library

1-(1) Extraction of Poly(A)⁺ RNA from ConA-Stimulated Rat Lymphoblasts

ConA-stimulated lymphoblasts (ConA blast) derived from rat spleen (about1×10⁶ cells/ml) were centrifuged (2,000×g) at 4° C. for 5 minutes. Theprecipitated cells were suspended with ISOGEN (Nippon Gene) andextracted with chloroform with shaking to collect the supernatant. Afterisopropanol was added to the obtained supernatant, the mixture wasallowed to stand at room temperature for 10 minutes and centrifuged at12,000×g at 4° C. for 10 minutes to precipitate RNA. The precipitatedRNA was washed with ethanol and dissolved in TE buffer. Poly(A)⁺ RNA waspurified from the total RNA so obtained with “mRNA Purification Kit”(Pharmacia).

1-(2) Preparation of cDNA

With 5 μg of the poly(A)⁺ RNA prepared above as a template, cDNA wassynthesized with “Time Saver cDNA Synthesis Kit” (Pharmacia). “Oligo dTprimer” (Pharmacia) having NotI site was used to increase the efficiencyof screening. EcoRI adapter was added, and digestion with NotI wasperformed to obtain cDNA with unidirectionality. Size fractionation wasthen performed with Spun Column (Pharmacia).

1-(3) Insertion Into a Vector

The obtained cDNA having EcoRI- and NotI-ends was ligated with pME18S(Hara et al., EMBO J., 11:1875-1884, 1992) digested with EcoRI and NotI.“DNA Ligation Kit” (Takara Shuzo) was used for the ligation. E. coli DH5cells (Toyobo) were transformed with the reaction product so obtained.Transformants were cultivated until O.D. value (at 600 nm) reached 0.6and harvested to recover plasmid DNAs with a library. QUIAGEN-Tip(QUIAGEN) was used for purification of plasmid DNAs.

2. Screening of cDNA Library

Screening was performed according to panning method (Seed et al., Proc.Natl. Acad. Sci. USA, 84:3365-3369, 1987).

2-(1) Gene Transfer into COS Cells

The library so obtained was introduced into COS7 cells byelectroporation (Potter et al., Proc. Natl. Acad. Sci. USA,85:2288-2292). The transformants were cultivated for 60 hours afterintroduction, the supernatant was removed, and the pellet was washedwith PBS three times. After the pellet was treated with PBS (containing0.5 mM EDTA) at 37° C. for 30 minutes, the cells were removed bypipetting. Only living cells were then collected with “Lymphprep”(NYCOMED).

2-(2) Concentration of Gene-Expressing Cells by Panning

The living cells obtained above were suspended in PBS (containing 5% FCSand 0.5 mM EDTA). The cell suspension was transferred to a culture dishcoated with “JTT-1 antibody” and incubated at room temperature for 3hours. After cells not binding to the culture dish were removed and theculture dish was washed with PBS three times, plasmid DNAs werecollected from the cells binding to the culture dish by Hirt method(Hirt, J. Mol. Biol., 26:365-369). E. coli DH10B (GIBCO BRL) weretransformed with the plasmid DNA so obtained. The plasmid DNAs wereamplified and purified with the transformants as in (1)-3 mentionedabove. The procedures described in (1) and (2) were then repeated twice.

2-(3) Isolation of the Positive Clone

After the third panning, transformed E. coli DH10B cells were cultivatedovernight on LB plates containing ampicillin to obtain colonies. Twentydrug-resistant colonies were cultivated, plasmid DNAs were collected byalkaline miniprep method (Maniatis et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.), and the insert DNA was analyzed. Agarose gel electrophoresisrevealed that the clone having about 0.9 kb cDNA (designated “T132A7”)was concentrated.

“T132A7” was transiently expressed in COS7 cells again with the methoddescribed in (1). After “T132A7”-introduced cells were reacted with“JTT-1 antibody” or “JTT-2 antibody”, and then with FITC-labeledanti-mouse IgG (Cappel), the fluorescence intensity of the stained cellswas measured with EPICS-Elite flow cytometer (Coulter). “JTT-1 antibody”and “JTT-2 antibody” strongly recognized the “T132A7” gene product. Theresults are shown in FIG. 8.

3. Determination of the Nucleotide Sequence and the Amino Acid Sequence

The nucleotide sequence of clone “T132A7” was determined by dideoxymethod with “Auto Read Sequencing Kit” (Pharmacia) and “A.L.F. DNASequencer” (Pharmacia). In addition, the deduced amino acid sequence of“rat JTT-1 antigen” encoded by the nucleotide sequence was analyzed withgene analysis software “GENEWORKS” (IntelliGenetics). The nucleotidesequence and the deduced amino acid sequence were shown in SEQ ID NO: 4and SEQ ID NO:13, respectively.

The amino acid sequence (composed of 200 amino acid residues) deducedfrom the cloned gene comprises the same amino acid sequence as the Nterminal amino acid sequence determined in Example 6-(3). Consideringthat clone “T132A7”-introduced cells strongly react with “JTT-1antibody,” it can be concluded that clone “T132A7” comprises the cDNAencoding “rat JTT-1 antigen.”

4. Computer Analysis

Hydropathy analysis of the primary structure of the deduced amino acidsequence of “JTT-1 antigen” was performed according to the method ofKite and Doolittle (Kite et al., J. Mol. Biol., 157:105-132, 1982) (FIG.9). The results revealed that “JTT-1 antigen” is a transmembrane proteinhaving a signal sequence at the N-terminus. In addition, the results ofmotif analysis revealed that “JTT-1 antigen” has two Asn-linked sugarchain binding sites in the extracellular domain, and two casein kinasephosphorylation sites and one protein kinase C phosphorylation site inthe cytoplasmic domain. In FIG. 9 “CHO” means N-linked sugar chainbinding site; “P”, phosphorylation site; “CKII”, casein kinase II; and“PKC”, protein kinase C.

Example 8 Cloning of cDNA Encoding “Human JTT-1 Antigen”

1. Preparation of a Probe

The cDNA (about 0.9 kb) encoding “rat JTT-1 antigen” was generated bydigesting the clone “T132A7” obtained in Example 7 with restrictionenzymes EcoRI and NotI, and separated by agarose gel electrophoresis.The separated DNA fragments were purified with “QUIAEX gel extractionkit” (QUIAGEN), and the obtained DNA fragments were labeled with ³²Pusing “Ready-To-Go DNA labelling kit” (Pharmacia). These labeled DNAfragments were used as probes for plaque hybridization.

2. Preparation of cDNA Library

2-(1) Extraction of Poly(A)⁺ RNA

Poly(A)⁺ RNA was extracted from ConA-stimulated lymphoblasts (ConAblast) derived from human peripheral blood in the same manner as inExample 7-1-(1).

2-(2) Preparation of cDNA

With 5 μg of the poly(A)⁺ RNA so prepared as a template, cDNAs weresynthesized with “oligo dT primer” (Pharmacia) and “Time Saver cDNASynthesis Kit” (Pharmacia). EcoRI adapter was then added, and sizefractionation was performed with Spun Column (Pharmacia).

2-(3) Insertion Into a Vector and Packaging

The cDNAs so obtained having EcoRI-ends were ligated with the vector“λZAPII” (Stratagene) digested with EcoRI. “DNA Ligation Kit” (TakaraShuzo) was used for the ligation. After in vitro packaging of theligated DNA was performed with “GIGA PACκ II GOLD” (Stratagene), E. coliXL1Blue MRF′ cells (Stratagene) were transfected with the obtained phageparticle to generate a cDNA library composed of plaque comprisingrecombinant phage.

3. Screening of cDNA Library

cDNA library was screened by plaque hybridization method (Maniatis etal., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) with “Rapid hybridization buffer”(Amersham). First, the cDNA library so obtained (1×10⁴) was plated ontoagar plates and the replica was produced with “Hybond-N nylon membrane”(Amersham). Plaque hybridization was performed in “Rapid hybridizationbuffer” (Amersham) using the replica and the ³²P-labeled probe preparedin Example 8-1. First and second screenings were performed to obtaineight positive clones. Single plaques of each clone were isolated andsubjected to in vivo excision in accordance with the manual (Stratagene)and seven positive clones were collected as plasmid DNA.

4. Determination of the Nucleotide Sequence

The nucleotide sequences of the seven clones were determined by dideoxymethod with “Auto Read Sequencing Kit” (Pharmacia) and “A.L.F. DNASequencer” (Pharmacia). The seven clones comprise the same nucleotidesequence. It was found that clone “pBSh4l” encodes the full length“human JTT-1 antigen.” The cDNA sequence corresponding to the openreading frame (ORF) of “human JTT-1 antigen” is shown in SEQ ID NO: 1,the full length of the deduced amino acid sequence of “human JTT-1antigen” is shown in SEQ ID NO: 2, and the nucleotide sequencecomprising 5′ and 3′ sequences is shown in SEQ ID NO: 3 (ORF correspondsto the nucleotide residues 26 to 625). It is understood that thenucleotide sequence contained in the clone encodes the full length of“human JTT-1 antigen” because the amino acid sequence (composed of 199amino acid residues) deduced from the nucleotide sequence showssignificant homology with the amino acid sequence of “rat JTT-1 antigen”(FIG. 10). As shown in FIG. 10, the homology between the amino acidsequences of human and rat “JTT-1 antigen” is 60% or more.

E. coli DH10B (GIBCO BRL) transformed with the clone “pBSh4l” has beendeposited under the Budapest Treaty with international depositoryauthority, National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, Ministry of International Tradeand Industry, Japan (1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) sinceOct. 25, 1996 (an international deposit accession No. FERM BP-5725).

5. Structural Characteristics and Biological Function of “JTT-1 Antigen”

The results of motif search for the deduced amino acid sequence of“human JTT-1 antigen” in known human proteins revealed that “human JTT-1antigen” has structural similarity to “CD28” and “CTLA-4,” human-derivedcell membrane proteins belonging to the immunoglobulin superfamily,mentioned in detail above (FIGS. 11 and 12). As mentioned above, “CD28”and “CTLA-4” are extremely important molecules regulating the activationand inhibition of T cells in immune system.

The structural similarity is as follows

1. 20 or more amino acid residues including cysteine residues are highlyconserved.

2. Proline repeating sequence, “Pro-Pro-Pro (PPP)”, which is essentialas the ligand binding region in CD28 and CTLA-4, is conserved.

3. “Tyr-Xaa-Xaa-Met (YxxM)” (Xaa and x represents any amino acid)sequence essential as the signal transmitting region in CD28 and CTLA-4is conserved in the cytoplasmic region.

From the fact that the same structure with the specific structure of“CD28” and “CTLA-4”, which play an important role in regulation ofactivation of T cells that are main actor in immune mechanism, “JTT-1antigen” of the present invention is inferred to play an important rolelike those molecules in regulation of activation of lymphocytes such asT cells which are main actor in immune response.

Example 9 Cloning of cDNA Encoding “Mouse JTT-1 Antigen”

1. Preparation of a Probe

The cDNA (about 0.9 kb) encoding “rat JTT-1 antigen” was obtained bydigesting the clone “T132A7, ” cloned in Example 7, with restrictionenzymes EcoRI and NotI, and separated by agarose gel electrophoresis.The DNA fragments so separated were purified with “QUIAEX gel extractionkit” (QUIAGEN), and the DNA fragments were labeled with ³²P using“Ready-To-Go DNA labelling kit” (Pharmacia). These labeled DNA fragmentswere used as a probe for plaque hybridization.

2. Preparation of cDNA Library

2-(1) Extraction of Poly(A)⁺ RNA

As Example 7-1-(1), poly(A)⁺ RNAs were extracted from ConA-stimulatedlymphoblasts derived from mouse spleen (about 1×10⁶ cells/ml).

2-(2) Preparation of cDNA Library

With 5 mg of poly(A)⁺ RNAs prepared in the above as a template, cDNAswere synthesized with oligo dT primer (Pharmacia) and “Time Saver cDNASynthesis Kit” (Pharmacia). After EcoRI adapter was added to the cDNA,size fractionation was performed with Spun Column (Pharmacia).

2-(3) Insertion of cDNA Into a Vector and Packaging

The cDNA so obtained having EcoRI-ends was ligated with the vectorlZAPII (Stratagene) digested with EcoRI. “DNA Ligation Kit” (TakaraShuzo) was used for the ligation. After in vitro packaging of theligated DNA was performed with GIGA PACκ II GOLD (Stratagene), E. coliXL1Blue MRF′ cells (Stratagene) were transfected with the phage particleso obtained to generate a cDNA library composed of plaque comprisingrecombinant phage.

3. Screening of cDNA Library

Screening was performed by plaque hybridization method (Maniatis et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, New York) using Rapid hybridization buffer(Amersham).

The above-obtained cDNA library (1×10⁴) was plated onto agar plates andthe replica was produced using Hybond-N nylon membrane (Amersham).Plaque hybridization was performed in Rapid hybridization buffer(Amersham) using the replica and the ³²P-labeled probe prepared inExample 9-1. First and second screenings were performed to obtain fivepositive clones. After single plaque of each clone was isolated, in vivoexcision was performed in accordance with Instruction Manual(Stratagene) and five positive clones were collected as plasmid DNA.

4. Determination of the Nucleotide Sequence

The nucleotide sequences of each of the five clones were determined bydideoxy method with “Auto Read Sequencing Kit” (Pharmacia) and “A.L.F.DNA Sequencer” (Pharmacia). The four of the five clones comprise thesame nucleotide sequence. The nucleotide sequence of cDNA encoding thefull length of “mouse JTT-1 antigen” and the deduced amino acid sequenceare shown in SEQ ID NO: 5 and SEQ ID NO:14, respectively.

As understood from FIG. 10, “mouse JTT-1 antigen” is composed of 200amino acid residues like “rat JTT-1 antigen.” The homology among theamino acid sequences of mouse, rat, and human “JTT-1 antigens” issignificant (60% or more).

5. Analysis of the Locus of “Mouse JTT-1 Antigen” Gene

The locus of the gene encoding “mouse JTT-1 antigen” was analyzed byfluorescence in situ hybridization method.

The cDNA so obtained encoding “mouse JTT-1 antigen” was labeled with ³²Pto prepare hybridization probes by the usual method. Using these probes,the 129 SVJ mouse genomic DNA library (Stratagene) was screened toobtain mouse genomic DNA clones comprising the exons encoding “mouseJTT-1 antigen.” The structure of the genomic DNA is schematically shownin FIG. 13.

The above-obtained genomic DNA clones were labeled with digoxigenin dUTPby nick translation to prepare probes. The labeled probes were bound tocleaved mouse DNA and hybridized with normal metaphase chromosomesderived from mouse embryonic fibroblasts in the solution containing 50%formaldehyde, 10% dextran sulfate, and 2×SSC. After a slide glass forhybridization was incubated in fluorescence-labeled anti-digoxigeninantibody, specific hybridization signal was detected by staining withDAPI. In the first test, the part near the largest chromosome that wasthought to be the chromosome 1, judging from the DNA size and emergedband, was specifically labeled. Based on this information, theabove-described genomic DNA clone was co-hybridized with probes specificto the centromere region of the chromosome 1. As a result, thecentromere region of the chromosome 1 and the regions near them werespecifically labeled. Ten samples of the chromosome 1 showing thespecific hybridization were analyzed, and it was revealed that theabove-mentioned genomic DNA clone was located at the position of 33% ofthe distance from the border between heterochromatin and euchromatin tothe telomere of the chromosome 1, namely, on the same band “1C3” as theloci of mouse “CD28” and “CTLA-4” genes. As the result that 80 metaphasecells were analyzed, specific labeling was identified at said positionfor 79 cells.

These results and the results obtained in Example 8 indicating thestructural similarity of “JTT-1 antigen” to “CD28” and “CTLA-4” suggestthat “JTT-1 antigen,” like “CD28” and “CTLA-4, ” is an importantmolecule involved in the regulation of the transmission of costimulatorysignal and/or activation of lymphocytes.

Example 10 Cloning of cDNA Encoding a Mutant of “Rat JTT-1 Antigen”

Another cDNA that is thought to encode alternative splicing variant of“rat JTT-1 antigen” cloned in Example 7 was cloned as follows.

1. Preparation of a Probe

The cDNA (about 0.9 kb) encoding “rat JTT-1 antigen” was generated bydigesting the clone “T132A7, ” obtained in Example 7, with restrictionenzymes EcoRI and NotI, and separated by agarose gel electrophoresis.The separated DNA fragments were purified with “QUIAEX gel extractionkit” (QUIAGEN), and the obtained DNA fragments were labeled with ³²Pusing “Ready-To-Go DNA labeling kit” (Pharmacia). These labeled DNAfragments were used as probes for plaque hybridization.

2. Preparation of cDNA Library

2-(1) Extraction of Poly(A)⁺ RNA

As in Example 7-1-(1), poly(A)⁺ RNA was extracted from rat thymoma cellline FTL435 (about 1×10⁶ cells/ml).

2-(2) Preparation of cDNA Library

With 5 mg of the poly(A)⁺ RNA prepared as mentioned above as a template,cDNAs were synthesized using oligo dT primer (Pharmacia) and “Time SavercDNA Synthesis Kit” (Pharmacia). After EcoRI adapter was added to thecDNA, size fractionation was performed with Spun Column (Pharmacia).

2-(3) Insertion of cDNA Into a Vector and Packaging

The cDNA having EcoRI-end obtained above was ligated with the vectorlZAPII (Stratagene) digested with EcoRI. “DNA Ligation Kit” (TakaraShuzo) was used for ligation. After in vitro packaging of the ligatedDNA was performed with GIGA PACκ II GOLD (Stratagene), E. coli XL1BlueMRF′ (Stratagene) was transfected with the obtained phage particle togenerate a cDNA library composed of plaque comprising recombinant phage.

3. Screening of cDNA Library

Screening was performed by plaque hybridization method (Maniatis et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.) with Rapid hybridization buffer (Amersham).

The above-prepared cDNA library (1×10⁴) was plated onto agar plates andthe replica was produced with Hybond-N nylon membrane (Amersham). Plaquehybridization was performed in Rapid hybridization buffer (Amersham)using the replica and the ³²P-labeled probe prepared in Example 10-1.First and second screenings were performed to obtain two positiveclones. After single plaque of each clone was isolated, in vivo excisionwas performed in accordance with Instruction Manual (Stratagene), andtwo positive clones were collected as plasmid DNA.

4. Determination of the Nucleotide Sequence

The nucleotide sequences of the two clones were determined by dideoxymethod with “Auto Read Sequencing Kit” (Pharmacia) and A.L.F. DNASequencer (Pharmacia). The two clones comprise the same nucleotidesequence. The nucleotide sequence of cDNA encoding the full length ofthe obtained “rat JTT-1 antigen” and the deduced amino acid sequence areshown in SEQ ID NO: 6 and SEQ ID NO:15, respectively. The amino acidsequence (SEQ ID NO: 6 or SEQ ID NO:15)deduced from the obtained cDNAsequence was compared with the amino acid sequence (SEQ ID NO: 4 or SEQID NO:13) deduced from the obtained cDNA sequence encoding “rat JTT-1antigen” cloned in Example 7 (FIG. 14). As shown in FIG. 14, the aminoacid sequence encoded by the cDNA cloned in this test was completely thesame as that encoded by the cDNA encoding “rat JTT-1 antigen” obtainedin Example 7, except that (1) C-terminal three continuous amino acidresidues (Met-Thr-Ser) changes into Thr-Ala-Pro, and that (2) subsequentto the Thr-Ala-Pro, 16 continuous amino acid residues (Leu-Arg-Ala-Leu-Gly-Arg-Gly-Glu-His-Ser-Ser-Cys-Gln-Asp-Arg-Asn) (SEQ ID NO:24) areadded. This indicates that the cDNA cloned in this test encodes thealternative splicing variant of “rat JTT-1 antigen” obtained in Example7

Example 11 Preparation of Recombinant “Human JTT-1 Antigen”-ExpressingCells

The plasmid clone pBSh4l obtained in Example 8 was digested with arestriction enzyme EcoRI, and a DNA fragment comprising the cDNAencoding the full length of “human JTT-1 antigen” was excised. This DNAfragment was inserted with DNA Ligation Kit (Takara Shuzo) into aplasmid pEFneo (Proc. Natl. Acad. Sci. USA 91:158-162, 1994) treatedwith the same restriction enzyme EcoRI to prepare the expression vector.CHO-K1 cells (ATCC: CCL-61) were transformed with the vector byelectroporation. By cultivating the cells in RPMI1640 medium containing0.8 mg/ml Geneticin (GIBCO BRL) and 10% fetal calf serum for about twoweeks, Geneticin-resistant transformants were selected. The expressionof recombinant “human JTT-1 antigen” was confirmed by Northern blottingby the usual method.

Example 12 Preparation of Monoclonal Antibodies Against “Human JTT-1Antigen”

The recombinant “human JTT-1 antigen”-expressing transformants preparedin Example 11 were homogenized and ultracentrifuged (100,000×g). Thepellet containing the cell membrane fraction was collected and suspendedin PBS. The resulting suspension comprising the cell membrane fractionwas injected into the footpad of a BALB/c mouse with complete Freund'sadjuvant for the first immunization (day 0). The cell membrane fractionantigen was further administered into its footpad at intervals of 7, 14,and 28 days. Two days after the last immunization, the lymph node cellswas taken out. The lymph node cells and mouse myeloma cells PAI (JCR No.B0113; Res. Disclosure 217:155, 1982) were mixed at a ratio of 5:1, andfused using polyethyleneglycol 4000 (GIBCO) as a fusing agent to preparemonoclonal antibody-producing hybridomas. The hybridomas were screenedby cultivating them in HAT-containing ASF104 medium (Ajinomoto)supplemented with 10% fetal calf serum and aminopterin. The culturesupernatant of each hybridoma was reacted with the recombinant “humanJTT-1 antigen”-expressing transformants prepared in Example 11, and thefluorescence intensity of cells stained by reacting them withFITC-labeled anti-mouse IgG (Cappel) was measured with EPICS-ELITE flowcytometer to confirm the reactivity of the monoclonal antibody generatedin each culture supernatant to “human JTT-1 antigen.” It has beenconfirmed that 10 or more kinds of hybridomas producing monoclonalantibodies reactive to “human JTT-1 antigen” were obtained.

Each of two kinds (designated clone SA12 and SG430) among thesehybridomas (10⁶ to 10⁷ cells/0.5 ml/mouse) was injected into a ICR nu/numouse (female, 7-8 weeks old) intraperitoneally. After 10 to 20 days,celiotomy of the mice was performed under anesthesia, and the two kindsof monoclonal antibodies (SA12 and SG430) reactive to “human JTT-1antigen” were prepared in a large amount from the ascites fluidextracted by the usual method.

Example 13 Effect of the Monoclonal Antibodies Against “Human JTT-1Antigen” on Human Peripheral Blood Lymphocytes

As mentioned in Example 8, it is thought that “JTT-1 antigen” can beinvolved in the regulation of the activation of lymphocytes in immunereaction like “CD28” and “CTLA-4. ” In order to prove this, the effectof the monoclonal antibodies against “human JTT-1 antigen” on humanlymphocytes was analyzed in light of cell growth as an indication.

To each well of 96-well microtiter plate were added (1) either SA12 orSG430 (1 μg/ml), the monoclonal antibody against “human JTT-1 antigen”prepared in Example 12, or (2) a mixture of either monoclonal antibodySA12 or SG430 (1 μg/ml) with anti-CD3 monoclonal antibody OKT-3 (1μg/ml, Orthodiagnostic Systems), which is used for adding the primarysignal in the activation of lymphocytes. The plate was incubated at 37°C. for 1 hour to coat each well with the antibody. After the plate waswashed with RPMI1640 medium, normal human peripheral blood lymphocytes(1×10⁵ cells/well) were added to each well and incubated in RPMI1640medium containing 10% fetal calf serum for 3 days. If necessary, 1 ng/mlphorbol myristate acetate (PMA) was added. Then, [³H] thymidine (3.7μkBq/well) was added to each well, and the plate was incubated at 37° C.for 6 hours. The cells were harvested, and the amount of [³H] thymidineincorporated into DNA was measured with a liquid scintillation counter(Beckman). The assay without any antibody was used as a control. Theresults are shown in FIG. 15.

In the assay using the plates coated with either monoclonal antibodySA12 or SG430, the number of lymphocytes increased about 10 timescompared to the control. In the co-presence of OKT3, the number oflymphocytes increased about 100 times when either monoclonal antibodySA12 or SG430 was used.

These results indicate that “JTT-1 antigen” functions in the regulationof the lymphocyte activation. The fact that the cell growth rate wasincreased by using together with OKT3 indicates that “JTT-1 antigen” isinvolved in the transmission of costimulatory signal like “CD28” and“CTLA-4.”

Example 14 Effect of “JTT-2 Antibody” on Experimental AllergicEncephalomyelitis (EAE)

As above mentioned in detail, recently, many attempts to treat variousautoimmune diseases (rheumatoid arthritis, multiple sclerosis,autoimmune thyroiditis, allergic contact dermatitis, chronicinflammatory dermatosis such as lichen planus, systemic lupuserythematosus, insulin dependent diabetes mellitus, psoriasis, etc.)have been made by regulating the transmission of between CD28/CTLA-4 andCD80/CD86. The effect has been already confirmed in various modelanimals of autoimmune diseases ((1) a model for human systemic lupuserythematosus (SLE); (2) experimental allergic encephalomyelitis (EAE),a model for multiple sclerosis (MS); (3) a model for insulin dependentdiabetes mellitus (IDDM); (4) Goodpasture's nephritis model; and (5)human rheumatoid arthritis).

In order to determine whether “JTT-1 antigen” of the present inventionis a molecule involved in the activation or inhibition of lymphocytessuch as “CD28” and “CTLA-4, ” model rats for experimental allergicencephalomyelitis (EAE), a model for multiple sclerosis (MS), wereproduced, and the effect of the titled monoclonal antibody on “JTT-1antigen” in the model was analyzed.

An emulsion to be used as immunogen was prepared by mixing Hartleyguinea pig cerebrospinal homogenate (800 mg/ml physiological saline)with the same amount of Freund's complete adjuvant. Immunization wasperformed by intradermally injecting the emulsion into left and rightfoot pads of 15 Lewis rats (female, 6-week-old) in an amount of 0.25 mlper footpad. The administration (immunization) was adjusted so as forthe dosages of the homogenate prepared to be 200 mg per rat. Thisimmunization so induces experimental allergic encephalomyelitis (EAE).

The rats so immunized were divided into three groups of five rats each,and any one of (1) to (3) below was intravenously injected into mice ofeach group immediately after immunization (day 0), and 3, 6, 9, and 12days after the immunization.

(1) Monoclonal antibody “JTT-2 antibody” against “rat JTT-1 antigen”prepared in Example 2 (dosage: 2 mg/ml PBS, 5 mg/kg)

(2) Prednisolone, steroid agent (dosage: 4 mg/ml PBS, 10 mg/kg)

(3) Control antibody non-reactive to “rat JTT-1 antigen” (dosage: 2mg/ml PBS, 5 mg/kg)

Symptom was observed in the course of time after the immunization. Afterthe onset of EAE had been found, the degree of the symptom was estimatedby scoring the symptom based on the following criteria.

(Score 1) Disappearance of tension of a tail

(Score 2) Dragging of hind legs, and slight paralysis

(Score 3) Dragging of hind legs, and serious paralysis

(Score 4) Paralysis of the whole body, or death

The results are shown in FIG. 16. In the group to which the controlantibody was administered, the symptom of EAE reached the peak (maximumscore) at day 11 to 15 after the immunization, and then graduallyrecovered. In contrast, in the “JTT-2 antibody”-administered group, thesymptom of EAE at day 11 after the immunization was significantlyinhibited. This inhibitory effect was significantly higher than that inthe prednisolone-administered group.

These results indicate that “JTT-1 antigen” is a molecule that functionsin the induction of immune response such as the lymphocyte activationinduced by immunization by foreign antigens, and that the regulation ofthe function of “JTT-1 antigen” or its ligands can inhibit the symptomof various autoimmune diseases.

Example 15 Effect of “JTT-2 Antibody” on Glomerulonephritis

For the same purpose as Example 14, glomerulus basement membrane (GBM)nephritis model rats were produced, and the effect of the titledmonoclonal antibody on “JTT-1 antigen” in the model was analyzed.

After bovine glomerulus basement membrane (Shigei Medical Institute)digested with collagenase was diluted with physiological saline to 200μg/ml, the dilution was mixed with Freund's complete adjuvant to preparean emulsion to be used as immunogen. Immunization was performed byintradermally injecting the emulsion into both hind soles of 48 Wistarkyoto rats (about 200 g) under anesthesia in an amount of about 0.2 mlper footpad (dosage: about 15 μg). This immunization so inducesglomerulus basement membrane (GBM) nephritis.

The immunized rats were divided into eight groups of six rats each, andany one of (1) to (3) below was injected into rats of each groupimmediately after immunization (day 0), and three times a week for 5consecutive weeks.

(1) Monoclonal antibody “JTT-2 antibody” against “rat JTT-1 antigen”prepared in Example 2 (dosage: 3 mg/kg (2 ml PBS/kg), intravenousinjection)

(2) Prednisolone, steroid agent, as a positive control (suspended in0.5% carboxymethylcellulose (CMC)) (dosage: 3 mg/kg (5 ml/kg), oraladministration)

(3) 0.5% CMC as a negative control (dosage: 5 ml/kg, oraladministration)

After the administration of a test substrate, sterilized water (25ml/kg) was orally administered into each rat forcedly, and urine wascollected for 5 hours from each rat which had been kept in a metabolismcage without eating and drinking. After the volume of the collectedurine was measured, the urinary protein concentration was measured usingTonein TP-II (Otuka), and the urinary excretion of protein per fivehours was calculated (unit: mg protein/5 hours). The above-mentionedurinary collection and urinary protein measurement were performed in thesame manner at 1, 2, 3, and 4 weeks after the immunization (day 0).

The results are shown in FIG. 17. Compared to the control group, theurinary excretion of protein at 3 weeks after the immunization wassignificantly reduced in the “JTT-2 antibody”-administered group.

These results indicate that “JTT-1 antigen” is a molecule that inducesimmune response such as the lymphocyte activation induced byimmunization by foreign antigens, and that the regulation of thefunction of “JTT-1 antigen” or its ligands can inhibit the symptom ofvarious autoimmune diseases.

Example 16 Preparation of the Fusion Protein Between “JTT-1 Antigen” andIgFc

As mentioned in Examples 8, and 13 to 15, “JTT-1 antigen” of the presentinvention is thought to be a molecule such as “CD28” and “CTLA-4”involved in the transmission of costimulatory signal involved in theregulation of the activation of lymphocytes. In addition, as mentionedin Example 14, a fusion protein (CTLA-4-IgFc) composed of theextracellular domain of “CTLA-4” and the Fc region of humanimmunoglobulin IgG1 reportedly has therapeutic effects on variousautoimmune diseases. In this Example, a fusion protein composed of theextracellular region of “JTT-1 antigen” and human IgGFc was prepared asfollows in order to examine whether soluble JTT-1 antigen, likeCTLA-4-IgFc, could be applied to therapy of various autoimmune diseases.

(1) Preparation of the Fusion Protein Between “Rat JTT-1 Antigen” andHuman IgG1-Fc (rJTT-1-IgFc)

In order to amplify the cDNA encoding the extracellular region of “ratJTT-1 antigen” by PCR, 5′ primer having XhoI restriction site(5′-CTGCTCGAGATGAAGCCCTACTTCTCG-3′, SEQ ID NO: 7) and 3′having BamHIrestriction site (5′-ACCCTACGGGTAACGGATCCTTCAGCTGGCAA-3′, SEQ ID NO:8)at their terminus were designed and synthesized. Using cDNA clone“T132A7” obtained in Example 7 encoding the full length of “rat JTT-1antigen” as a template, PCR was performed with the primers to preparethe cDNA comprising the cDNA encoding the extracellular region of “ratJTT-1 antigen” having XhoI and BamHI restriction sites at its both ends.The PCR products so obtained were digested with XhoI and BamHI andseperated by agarose gel electrophoresis to isolate an about 450-bp bandpredicted to be the cDNA fragment encoding a desired extracellularregion. The isolated cDNA fragment was subloned into pBluescript II SK(+) (Stratagene) cleaved with XhoI and BamHI. Sequence analysis with anautomated flourescence DNA sequencer (Applied Biosystems) revealed thatthe cDNA fragment comprises the region encoding amino acid sequencecorresponding to the amino acid residues 1 to 141 of “rat JTT-1 antigen”(SEQ ID NO:4 or SEQ ID NO:13).

On the other hand, the DNA encoding the Fc of human IgG1 as the fusionpartner was cut out as an about 1.3 kb BamHI-XbaI DNA fragment bydigesting the plasmid (see Cell 61:1303-1313, 1990). Prepared by B. Seedet al. (Massachusetts General Hospital)) with BamHI and XbaI. Thisfragment comprises exons encoding human IgG1 hinge region, Cη₁2, andCη₁3.

The XhoI-BamHI fragment encoding the extracellular region of “rat JTT-1antigen,” and BamHI-XbaI fragment comprising exons encoding the Fc ofhuman IgG1 (“IgFc”), both prepared as mentioned above, were subclonedinto pBluescript II SK (+) (Stratagene) cleaved with XhoI and XbaI.

Then, the plasmid was digested with XhoI and XbaI, and an about 1.8 kbDNA fragment comprising the fusion DNA comprising the extracellularregion of “rat JTT-1 antigen” and human IgFc was cut out. This fusionDNA fragment was inserted into the XhoI and XbaI sites of the expressionvector pME18S (Medical Immunology 20:27-32, 1990; Experimental Medicine:SUPPLEMENT, “Handbook of Genetic Engineering,” Yodosha, pp. 101-107,1992) with T4 DNA ligase to construct plasmid prJTT-1-IgFc.

HEK293 cells (ATCC CRL1573) subconfluently cultivated as monolayer inDMEM medium containing 10% fetal calf serum and ampicillin weretransformed with prJTT-1-IgFc by electroporation to obtaintransformants.

The transformants were cultured in serum-free ASF104 medium for 72 hoursto express rJTT-1-IgFc.

Using a Protein G Sepharose affinity column (Pharmacia), rJTT-1-IgFc waspurified as follows.

The supernatant obtained by centrifuging the culture medium mentionedabove was loaded onto Protein G Sepharose affinity column previouslyequilibrated with binding buffer. After the column was washed withbinding buffer, elution was performed with elution buffer. The eluatewas collected and dialyzed against phosphate buffer with exchanging theexternal solution twice or more to obtain pure rJTT-1-IgFc.

The result of affinity chromatography is shown in FIG. 18, and theresult of SDS-PAGE of the pure rJTT-1-IgFc so obtained in FIG. 19.

(2) Preparation of the Fusion Protein Between “Human JTT-1 Antigen” andHuman IgG1-Fc (hJTT-1-IgFc)

hJTT-1-IgFc was prepared as mentioned above in (1), except for cDNA usedas templates and primers for PCR. In this test, the clone “pBSh41”comprising the cDNA encoding the full length “human JTT-1 antigen”prepared in Example 8 was used as a template, and5′-TAACTGTTTCTCGAGAACATGAAGTCAGGC-3′ (SEQ ID NO: 9) and5′-ATCCTATGGGTAACGGATCCTTCAGCTGGC-3′ (SEQ ID NO: 10) were used asprimers.

The result of affinity chromatography is shown in FIG. 20, and theresult of SDS-PAGE of the pure hJTT-1-IgFc so obtained in FIG. 21.

Example 17 Preparation of a Transgenic Mouse in Which cDNA Encoding “RatJTT-1 Antigen” has Been Integrated

The cDNA encoding the full length of “rat JTT-1 antigen” obtained inExample 7 was inserted into the expression vector pCAGGS (Gene108:193-200, 1991) having chicken β actin promoter using DNA BluntingKit (Takara) to obtain plasmid, prJTT-1. In order to prepare atransgenic mouse, prJTT-1 was linearized by restriction enzymetreatment.

A female ICR mouse having a vaginal plug, obtained by mating a white ICRmouse (Nihon LSC) with a male vasoligated white ICR mouse (Nihon SLC),was used as a foster mother mouse. A mouse for obtaining fertilized eggsfor introducing “rat JTT-1 antigen” gene thereinto was prepared bymating a female BDF-1 mouse (Nihon SLC) that had been made tosuperovulate by administered PEAMEX (5 units, Sankyo Zoki) and Pregnil(5 units, Organon) with a male BDF-1 male (Nihon SLC). After mating, theoviduct was excised from the female BDF-1 mouse, and only fertilizedeggs were obtained by hyaluronidase treatment and stored in a medium.

The “rat JTT-1 antigen” gene was introduced into the fertilized eggunder microscopy using a manipulator according to the usual method. Thefertilized egg was fixed with a retaining needle. A solution containingthe above-mentioned linearized gene encoding “rat JTT-1 antigen,” whichwas diluted with Tris-EDTA buffer, was microinjected into the malepronucleus of the fertilized eggs with a DNA introduction needle at 37°C.

After gene introduction, only fertilized eggs keeping normal state wereselected, and then, the fertilized egg so selected in which the “ratJTT-1 antigen” genes have been introduced was inserted into the ovarianfimbria in the ovary of a foster mother mouse (white ICR mouse).

The tail of a progeny mouse (founder mouse) born from the foster mothermouse was cut off and the genomic gene was collected from it. It wasconfirmed by PCR that the “rat JTT-1 antigen” gene was integrated intothe mouse genome. Then, heterozygous transgenic mice highly expressing“rat JTT-1 antigen” were prepared by mating this founder mouse with anormal mouse. Homozygous transgenic mice can be prepared by mating theheterozygous mice with each other.

The microinjected construct comprising the “rat JTT-1 antigen” gene isschematically shown in FIG. 22.

Example 18 Preparation of a Knockout Mouse Whose Endogenous GeneEncoding “Mouse JTT-1 Antigen” has Been Inactivated

(1) Construction of a Targeting Vector

A targeting vector for inactivating (knocking out) the endogenous geneencoding “mouse JTT-1 antigen” through homologous recombination (NikkeiScience, pp. 52-62, May 1994) was prepared as follows.

The PstI-HindIII fragment (“homologous DNA (1)”) obtained by digestingthe mouse genomic DNA clone comprising the region encoding “mouse JTT-1antigen” cloned in Example 9-5 with PstI and HindIII was subcloned intopGEM-3 (Promega). Then, pGEM-3 was linearized with XhoI, and neomycinresistance gene (“neo”) excised from pMC1-neo-polyA (Stratagene) bytreating it with XhoI and SalI was inserted at the upstream of the“homologous DNA” (1) and then ligated them. The above-mentioned mousegenomic DNA clone was digested with XhoI and NotI to cut off an about5.5 kb gene (“homologous DNA (2)”) located upstream of above-mentioned“homologous DNA (1).” Separately, the above-mentioned pGEM-3 into which“neo-homologous DNA (1)” has been inserted was digested with XhoI andHindIII to cut off “neo-homologous DNA (1).” “Homologous DNA (2)” and“neo-homologous DNA (1)” thus obtained were subcloned into pSEAP2-CONT(Clontech) linearized with NotI and HindIII.

After the obtained plasmid, in which “homologous (2)-neo-homologous (1)”has been inserted, was digested and linearized at the downstream of“homologous DNA (1) with NruI, thymidine kinase gene (“TK”) obtained bydigesting pMCl-TK (Stratagene) with PvuII was inserted at the downstreamof “homologous DNA (1)” to obtain a targeting vector, in which“homologous DNA (2)-neo-homologous (1)” was inserted.

(2) Introduction of the Targeting Vector into ES Cells

Mouse embryonic stem cells (Nature 362:255-258, 1993; Nature326:292-295, 1987) cultured in DMEM medium containing 15% fetal calfserum were treated with trypsin to be single cells, and the cells werewashed three times, followed by adding phosphate buffer thereto toadjust 1×10⁷ cells/ml. The targeting vector mentioned above (25 μg per 1ml of the cell suspension) was added to the cell suspension, andelectric pulse was delivered once under the condition of 350 V/cm (25μF). Then, 1×10⁷ of ES cells were plated on a 10-cm dish and cultivatedin maintenance medium for a day, and the medium was changed to selectionmedium (containing 250 μg/ml G418 and 2 μM ganciclovir). The cells werecultivated with the medium changed every two days. At the tenth day fromthe introduction of the targeting vector, 573 neomycin-resistant ES cellclones were obtained under microscopy with a micropipet. Each of the EScell clones so obtained were cultivated independently on a 24-well platecoated by Feeder cells to obtain 768 neomycin-resistant ES cellreplicas.

(3) Screening of Knockout ES Cells

It was confirmed by PCR whether the endogenous gene encoding “mouseJTT-1 antigen” was disrupted (knocked out) through homologousrecombination in each of the neomycin-resistant ES cells.

For PCR, (1) primers designed and synthesized based on the sequence ofabove-mentioned neomycin-resistant gene (“neo”)(5I-CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGC-3′, SEQ ID NO: 11) and (2)primers designed and synthesized based on the sequence ofabove-mentioned “homologous DNA (1)”(5′-CATTCAAGTTTCAGGGAACTAGTCCATGCGTTTC-3′, SEQ ID NO: 12) were used.

Each genomic DNA was extracted from each of the neomycin-resistant EScell, and PCRs were performed using the primers with the genomic DNA asa template. PCR was performed 1 cycle of reaction at 94° C. for 3minutes, 30 cycles of reaction at 94° C. for 1 minute, at 60° C. for 1minute, and at 72° C. for 3.5 minutes, and 1 cycle of reaction at 72° C.for 10 minutes, and the resulting products were stored at 4° C. When afragment less than about 4 kb was amplified by this PCR, it could bejudged that the endogenous gene encoding “mouse JTT-1 antigen” wasdisrupted (knocked out) through homologous recombination in the ES cellclone.

A desired PCR product was obtained from three of 768 ES cell clonestested. Genomic southern blottings were performed for these three clonesfor further screening and confirmation. After genomic DNA was extractedfrom the three clones and digested with restriction enzyme BamHI, thedigested products were subjected to agarose gel electrophoresis. Theresulting DNAs were transferred to nylon membrane, and hybridization wasperformed with a probe prepared from the genomic DNA sequence comprising“mouse JTT-1.” The probe was designed based on the sequence outside thesite where homologous recombination occurred, which enables todistinguish mutant type genome from normal type genome in size.

As a result, two bands corresponding to mutant type and normal type wereobserved in one of the three clones. This ES cell clone was used for thepreparation of a knockout mouse described below.

(4) Preparation of a Knockout Mouse

The above-obtained ES cells (15 ES cells per blastocyst) whoseendogenous gene encoding “mouse JTT-1 antigen” has been inactivated(knocked out) through homologous recombination were microinjected intoblastocysts to, which were obtained by mating a female C57BL6 mouse(Nihon Charles River) with male one. Immediately after themicroinjection, the blastocysts (about 10 blastocysts per one side ofthe uterus) were transplanted in the uterus of a foster mother ICR mouse(CLEA Japan), which was 2.5 day-mouse from pseudopregnant treatment. Asa result, 38 progeny mice in total were obtained, and 18 out of themwere desired chimeric mice. Eleven (11) out of the chimeric mice werethe chimeric-mouse in which the contribution to hair color was 80% ormore.

The chimeric mice so obtained were then mated with a normal C57BL6 miceto obtain agouti mice whose color is derived from hair color gene of theES cells.

Example 19 Preparation of Pharmaceutical Composition Comprising Antibody

Each of the monoclonal antibody (50-150 μg/ml), “JTT-1 antibody” and“JTT-2 antibody” against “rat JTT-1 antigen,” prepared in Example 1, andmonoclonal antibodies, “SA12” and “SG430” against “human JTT-1 antigen,”prepared in Example 12, was added to injectable distilled water (10 ml)to prepare injection.

Industrial Applicability

Novel cell surface molecules (called “JTT-1 antigen”) of the presentinvention derived from mammals such as human, mouse, and rat arecharacterized as follows.

(1) “JTT-1 antigen” had the following similarity with “CD28,” a cellsurface molecule on lymphocytes such as T cells, which transmitscostimulatory signal important for T cell activation through celladhesion, and “CTLA-4,” a cell surface molecule on lymphocytes such as Tcells, which regulates the function of activated lymphocytes such asactivated T cells, cooperating with the signal.

-   -   (i) 20 or more amino acid residues including cysteine residues        are highly conserved;    -   (ii) Proline repeating sequence, “Pro-Pro-Pro (PPP),” which is        essential as the ligand binding region, is conserved in the        extracellular region;    -   (iii) “Tyr-Xaa-Xaa-Met (YxxM)” (Xaa and x represents any amino        acid) sequence essential as the signal transmitting region is        conserved in the cytoplasmic region; and    -   (iv) The locus of the gene encoding “mouse JTT-1 antigen” on        mouse chromosome is “1C3”, like “CD28” and “CTLA-4 .”

(2) “JTT-1 antigen” can mediate cell adhesion of thymocytes,lymphoblasts stimulated with mitogen such as ConA, thymomas, like “CD28”and “CTLA-4” that mediate cell adhesion.

(3) “JTT-1 antigen” is strongly expressed, at least, in thymocytes,lymphoblast cells stimulated with mitogen such as ConA (activated Tlymphoblast cells and activated B lymphoblast cells, etc.), peripheralblood lymphocytes, and thymomas.

(4) The antibody against “JTT-1 antigen” significantly proliferateshuman peripheral blood lymphocytes, and the proliferation is moreenhanced in the presence of a monoclonal antibody against CD3constituting TcR/CD3 complex on T cells that receive the primary signalessential for T cell activation from antigen-presenting cells.

(5) The administration of the antibody against “JTT-1 antigen”significantly inhibits the symptom of experimental allergicencephalomyelitis (EAE).

(6) The administration of the antibody against “JTT-1 antigen” to amodel rat for glomerulus basement membrane (GBM) nephritis significantlyinhibits the symptom of this disease.

“JTT-1 antigen” of the present invention is, like “CD28” and “CTLA-4,”thought to be a molecule transmitting the secondary signal(costimulatory signal) essential for the activation of lymphocytes suchas T cells, and regulating the function of activated lymphocytes such asactivated T cells, cooperating with the signal.

Therefore, polypeptides constituting such cell surface molecules, itspolypeptide fragment, and fusion polypeptides therefrom, and antibodiesthereto of the present invention can provide extremely usefulpharmaceuticals for therapy or prevention of various autoimmunediseases, allergic diseases, or inflammatory diseases, specifically,rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis,allergic contact dermatitis, chronic inflammatory dermatosis such aslichen planus, systemic lupus erythematosus, insulin dependent diabetesmellitus, and psoriasis, caused by the activation of lymphocytes such asT cells and the abnormality of regulation of activated lymphocytefunctions.

Similarly, the genes encoding polypeptides or polypeptide fragments ofthe present invention can be used in not only gene therapy of variousdiseases as mentioned above but also preparation of antisensepharmaceuticals.

Among the antibodies of the present invention, human monoclonalantibodies and their pharmaceutical compositions have dramaticallyincreased pharmaceutical value of antibody drugs because they have noantigenicity against human, which has been a serious problem (sideeffect) of antibody pharmaceuticals containing nonhuman mammal-derivedantibodies such as mouse-derived antibodies.

The genes (DNA), polypeptides, polypeptide fragments and antibodies ofthe present invention are useful not only as pharmaceuticals but also asreagents for searching molecules (ligands) interacting with the cellsurface molecules of the present invention, clarifying the function ofthe ligand, and developing drugs targeting the ligands.

Furthermore, the transgenic mouse of the present invention is extremelyuseful not only as a model animal for studying physiological function of“JTT-1 antigen” that is a cell surface molecule of the present inventionbut also as a tool for screening various drugs (low molecular weightcompounds, antibodies, antisense substances, polypeptides, etc.) havingactivity regulating (inhibition, suppression, activation, stimulation,etc.) the function of “JTT-1 antigen.” Specifically, such testsubstances can be administered to the transgenic mouse to measure andanalyze various physiological, biological, or pharmacological parametersgenerated in the mouse, thereby assessing activity of the administeredtest substances.

In addition, the knockout mouse of the present invention can clarify thefunction of the cell surface molecules of the present invention byanalyzing the characteristics of the mouse from various viewpoints(physiological, biological, pharmacological, pathological, and geneticviewpoints).

1. A method of inhibiting the transmission of a costimulatory signal oflymphocytes, the method comprising administering to a subject apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a polypeptide comprising (a) an extracellular region of thepolypeptide set forth in SEQ ID NO:2, or (b) an extracellular region ofa polypeptide that consists of the amino acid sequence of SEQ ID NO:2 inwhich one to ten amino acid residues are substituted, deleted or added;wherein said polypeptide comprises the amino acid sequencePhe-Asp-Pro-Pro-Pro-Phe (SEQ ID NO:21) and inhibits transmission of acostimulatory signal of lymphocytes.
 2. The method of claim 1, whereinthe polypeptide consists of (a) an extracellular region of thepolypeptide set forth in SEQ ID NO:2, or (b) an extracellular region ofa polypeptide that consists of the amino acid sequence of SEQ ID NO:2 inwhich one to ten amino acid residues are substituted, deleted or added.3. A method of inhibiting the transmission of a costimulatory signal oflymphocytes, the method comprising administering to a subject apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a polypeptide comprising amino acids 1-140 of SEQ ID NO:2.4. A method of inhibiting the transmission of a costimulatory signal oflymphocytes, the method comprising administering to a subject apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a polypeptide consisting of amino acids 1-140 of SEQ IDNO:2.
 5. A method of inhibiting the transmission of a costimulatorysignal of lymphocytes, the method comprising administering to a subjecta pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a homodimer molecule consisting of two polypeptide fragmentsbridged through disulfide bonds to each other, wherein each polypeptidefragment comprises the amino acid sequence Phe-Asp-Pro-Pro-Pro-Phe (SEQID NO:21) and comprises (a) an extracellular region of the polypeptideset forth in SEQ ID NO:2, or (b) an extracellular region of apolypeptide that consists of the amino acid sequence of SEQ ID NO:2 inwhich one to ten amino acid residues are substituted, deleted or added;wherein an antibody reactive with the homodimer molecule inducesproliferation of peripheral blood lymphocytes in the presence of anantibody reactive with CD3.
 6. The method of claim 5, wherein eachpolypeptide fragment comprises an extracellular region of thepolypeptide set forth in SEQ ID NO:2.
 7. The method of claim 5, whereineach polypeptide fragment consists of an extracellular region of thepolypeptide set forth in SEQ ID NO:2.
 8. The method of claim 5, whereineach polypeptide fragment consists of an extracellular region of apolypeptide that consists of the amino acid sequence of SEQ ID NO:2 inwhich one to ten amino acid residues are substituted, deleted or added.9. A method of inhibiting the transmission of a costimulatory signal oflymphocytes, the method comprising administering to a subject apharmaceutical composition comprising a pharmaceutically acceptablecarrier and fusion polypeptide comprising (a) a polypeptide consistingof an extracellular region of (i) the polypeptide set forth in SEQ IDNO:2, or (ii) a polypeptide that consists of the amino acid sequence ofSEQ ID NO:2 in which one to ten amino acid residues are substituted,deleted or added; and (b) a portion of a constant region of a humanimmunoglobulin heavy chain; wherein said fusion polypeptide comprisesthe amino acid sequence Phe-Asp-Pro-Pro-Pro-Phe (SEQ ID NO:21) andinhibits the transmission of a costimulatory signal of lymphocytes. 10.The method of claim 9, wherein the fusion polypeptide consists of (a) apolypeptide consisting of an extracellular region of (i) the polypeptideset forth in SEQ ID NO:2, or (ii) a polypeptide that consists of theamino acid sequence of SEQ ID NO:2 in which one to ten amino acidresidues are substituted, deleted or added; and (b) a portion of aconstant region of a human immunoglobulin heavy chain.
 11. The method ofclaim 9, wherein the extracellular region is amino acid residues 1-140of SEQ ID NO:2.
 12. The method of claim 9, wherein the portion of theconstant region of a human immunoglobulin heavy chain consists of thehinge region, CH2 domain, and CH3 domain of human IgG heavy chain. 13.The method of claim 11, wherein the portion of the constant region of ahuman immunoglobulin heavy chain consists of the hinge region, CH2domain, and CH3 domain of human IgG heavy chain.
 14. The method of claim10, wherein the extracellular region is amino acid residues 1-140 of SEQID NO:2.
 15. The method of claim 10, wherein the portion of the constantregion of a human immunoglobulin heavy chain consists of the hingeregion, CH2 domain, and CH3 domain of human IgG heavy chain.
 16. Themethod of claim 14, wherein the portion of the constant region of ahuman immunoglobulin heavy chain consists of the hinge region, CH2domain, and CH3 domain of human IgG heavy chain.
 17. A method ofinhibiting the transmission of a costimulatory signal of lymphocytes,the method comprising administering to a subject a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and ahomodimer molecule consisting of two fusion polypeptides bridged throughdisulfide bonds to each other, each fusion polypeptide comprising (a) apolypeptide consisting of an extracellular region of (i) the polypeptideset forth in SEQ ID NO:2, or (ii) a polypeptide that consists of theamino acid sequence of SEQ ID NO:2 in which one to ten amino acidresidues are substituted, deleted or added; and (b) a portion of aconstant region of a human immunoglobulin heavy chain; wherein eachfusion polypeptide comprises the amino acid sequencePhe-Asp-Pro-Pro-Pro-Phe (SEQ ID NO:21), and wherein the homodimerinhibits the transmission of a costimulatory signal of lymphocytes. 18.The method of claim 17, wherein each fusion polypeptide consists of (a)a polypeptide consisting of an extracellular region of (i) thepolypeptide set forth in SEQ ID NO:2, or (ii) a polypeptide thatconsists of the amino acid sequence of SEQ ID NO:2 in which one to tenamino acid residues are substituted, deleted or added; and (b) a portionof a constant region of a human immunoglobulin heavy chain.
 19. Themethod of claim 17, wherein the extracellular region is amino acidresidues 1-140 of SEQ ID NO:2.
 20. The method of claim 17, wherein theportion of the constant region of a human immunoglobulin heavy chainconsists of the hinge region, CH2 domain, and CH3 domain of human IgGheavy chain.
 21. The method of claim 19, wherein the portion of theconstant region of a human inimunoglobulin heavy chain consists of thehinge region, CH2 domain, and CH3 domain of human IgG heavy chain. 22.The method of claim 18, wherein the extracellular region is amino acidresidues 1-140 of SEQ ID NO:2.
 23. The method of claim 18, wherein theportion of the constant region of a human immunoglobulin heavy chainconsists of the hinge region, CH2 domain, and CH3 domain of human IgGheavy chain.
 24. The method of claim 22, wherein the portion of theconstant region of a human immunoglobulin heavy chain consists of thehinge region, CH2 domain, and CH3 domain of human IgG heavy chain.